Treatment of Rb-negative tumors using topoisomerase with cyclin dependent kinase 4/6 inhibitors

ABSTRACT

This invention is in the area of improved therapeutic combinations for and methods of treating selected retinoblastoma (Rb)-negative cancers and Rb-negative abnormal cellular proliferative disorders using particular topoisomerase inhibitors and specific cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. In one aspect, the improved treatment of select Rb-negative cancers is disclosed using specific compounds disclosed herein in combination with a topoisomerase I inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation U.S. application Ser. No. 15/457,667,filed Mar. 13, 2017, which is a continuation of International PatentApplication No. PCT/US2015/049756, filed Sep. 11, 2015, which claims thebenefit of U.S. Provisional Application No. 62/050,035 filed Sep. 12,2014. These applications are hereby incorporated by reference for allpurposes.

FIELD

This invention is in the area of improved therapeutic combinations forand methods of treating selected retinoblastoma (Rb)-negative cancersand Rb-negative abnormal cellular proliferative disorders usingparticular topoisomerase inhibitors and specific cyclin-dependent kinase4/6 (CDK4/6) inhibitors. In one aspect, the improved treatment of selectRb-negative cancers is disclosed using specific compounds disclosedherein in combination with a topoisomerase I inhibitor.

BACKGROUND

The regulation of the cell cycle is governed and controlled by specificproteins, which are activated and deactivated mainly throughphosphorylation/dephosphorylation processes in a precisely-timed manner.The key proteins that coordinate the initiation, progression, andcompletion of cell-cycle program are cyclin dependent kinases (CDKs).Cyclin-dependent kinases belong to the serine-threonine protein kinasefamily. They are heterodimeric complexes composed of a catalytic kinasesubunit and a regulatory cyclin subunit. CDK activity is controlled byassociation with their corresponding regulatory subunits (cyclins) andCDK inhibitor proteins (Cip & Kip proteins, INK4s), by theirphosphorylation state, and by ubiquitin-mediated proteolytic degradation(see D. G. Johnson, C. L. Walker, Annu. Rev. Pharmacol. Toxicol 39(1999) 295-312; D. O. Morgan, Annu. Rev. Cell Dev. Biol. 13 (1997)261-291; C. J. Sherr, Science 274 (1996) 1672-1677; T. Shimamura et al.,Bioorg. Med. Chem. Lett. 16 (2006) 3751-3754).

There are four CDKs that are significantly involved in cellularproliferation: CDK1, which predominantly regulates the transition fromG2 to M phase, and CDK2, CDK4, and CDK6, which regulate the transitionfrom G1 to S phase (Malumbres M, Barbacid M. Cell cycle, CDKs andcancer: a changing paradigm. Nat. Rev. Cancer 2009; 9(3):153-166). Inearly to mid G1 phase, when the cell is responsive to mitogenic stimuli,activation of CDK4-cyclin D and CDK6-cyclin D induces phosphorylation ofthe retinoblastoma protein (pRb). Phosphorylation of pRb releases thetranscription factor E2F, which enters the nucleus to activatetranscription of other cyclins which promote further progression of thecell cycle (see J. A. Diehl, Cancer Biol. Ther. 1 (2002) 226-231; C. J.Sherr, Cell 73 (1993) 1059-1065). CDK4 and CDK6 are closely relatedproteins with basically indistinguishable biochemical properties (see M.Malumbres, M. Barbacid, Trends Biochem. Sci. 30 (2005) 630-641).

A number of CDK 4/6 inhibitors have been identified, including specificpyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas,benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, andoxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer DrugTargets 8 (2008) 53-75). For example, WO 03/062236 identifies a seriesof 2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment ofRb positive cancers that show selectivity for CDK4/6, including6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one(PD0332991), which is currently being tested by Pfizer in late stageclinical trials as an anti-neoplastic agent against estrogen-positive,HER2-negative breast cancer. Tate, et al. describe the antitumoractivity of the CDK4/6 inhibitor abemaciclib (LY2835219)(“Semi-Mechanistic Pharmacokinetic/Pharmacodynamic Modeling of theAntitumor Activity of LY2835219, a New Cyclin-Dependent Kinase 4/6Inhibitor, in Mice Bearing Human Tumor Xenografts”, Clin Cancer Res(Jul. 15, 2014) 20; 3763). Rader, et al. describe the reducedproliferation in neuroblastoma-derived cell lines using the CDK4/6inhibitor ribociclib (LEE011) (“Dual CDK4/CDK6 Inhibition Induces CellCycle Arrest and Senescence in Neuroblastoma”, Clin Cancer Res (Nov. 15,2013) 19(22): 6173-82). VanderWel et al. describe an iodine-containingpyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005) 2371-2387). WO99/15500 filed by Glaxo Group Ltd discloses protein kinase andserine/threonine kinase inhibitors. WO 2010/020675 filed by Novartis AGdescribes pyrrolopyrimidine compounds as CDK inhibitors. WO 2011/101409also filed by Novartis describes pyrrolopyrimidines with CDK 4/6inhibitory activity. WO 2005/052147 filed by Novartis and WO 2006/074985filed by Janssen Pharma disclose additional CDK4 inhibitors. WO2012/061156 filed by Tavares and assigned to G1 Therapeutics describesCDK inhibitors. WO 2013/148748 filed by Francis Tavares and assigned toG1 Therapeutics describes Lactam Kinase Inhibitors. PCT PatentApplication No. PCT/US2014/029073 filed by Strum et al. and assigned toG1 Therapeutics describes compounds and methods for protection ofhematopoietic stem and progenitor cells against ionizing radiation usingCDK4/6 inhibitors. In one aspect, PCT/US2014/029073 describes the use ofa CDK4/6 inhibitor to protect hematopoietic stem and progenitor cells ina subject with small cell lung cancer undergoing treatment with standardof care chemotherapeutics such as carboplatin, cisplatin, etoposide,topotecan, camptothecin, and irinotecan. PCT Patent Application No.PCT/US2014/028685 filed by Strum et al. and assigned to G1 Therapeuticsdescribes compounds and methods for protection of normal cells duringchemotherapy using CDK4/6 inhibitors. PCT Patent Application No.PCT/US2014/029429 filed by Strum et al. and assigned to G1 Therapeuticsdescribes compounds and methods for treating Rb-positive cancers usingCDK4/6 inhibitors. PCT Patent Application No. PCT/US2014/029274 filed byStrum et al. and assigned to G1 Therapeutics describes compounds andmethods for treating certain cancers with CDK4/6 inhibitors.

Selective CDK4/6 inhibitors are generally designed to targetCDK4/6-replication dependent cancers. For example, Michaud et al.,reported that the CDK4/6 inhibitor PD-0332991 was inactive againstRb-negative tumors. (Michaud et al., Pharmacologic inhibition ofcyclin-dependent kinase 4 and 6 arrests the growth of glioblastomamultiform intracranial xenografts. Cancer Res. 70:3228-3238 (2010)).

Topoisomerase enzymes play a vital role in cellular proliferation andreplication, altering the supercoiling of double-stranded DNA bycatalyzing the breaking and rejoining of the phosphodiester backbone ofDNA strands during the normal cell cycle. DNA strand separation isobligatory to transcribe and replicate genomes by copying each base byRNA and DNA polymerases. (Pommier et al., DNA topoisomerases and theirpoisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17(2010) 421-433). Because of DNA's double-helical structure, replicationgenerated catenated progenies that have to be unlinked by topoisomerasesprior to cytokinesis.

Topoisomerases are classified as type I and type II. Type I enzymescleave one DNA strand at a time and type II both strands to performtheir catalytic functions. All topoisomerases cleave the DNAphosphodiester backbone by nucleophilic attack from a catalytic tyrosineresidue which becomes linked to the phosphate end (P-Y) of the DNAbreak. Those reactions are highly reversible and leave the DNA sequenceunchanged following topoisomerization (Pommier et al., DNAtopoisomerases and their poisoning by anticancer and antibacterialdrugs, Chem. & Biol. Review 17 (2010) 421-433).

A number of topoisomerase type I (Top1) inhibitors have been evaluatedas anticancer therapeutics. Camptothecin was first identified from theChinese tree Camptotheca acuminate (Wall et al., “The isolation andstructure of camptothecin, a novel alkaloidal leukemia and tumorinhibitor from Camptotheca acuminate,” J. Am. Chem. Soc. (1966) 88:3888-3890.) A number of camptothecin derivatives, including for exampletopotecan, irinotecan, belotecan, gimatecan, lurtotecan, diflomotecan,S39625, and exatecan, have been further investigated as anticanceragents (Pommier et al., DNA topoisomerases and their poisoning byanticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010)421-433).

In addition to the camptothecin derivatives, several non-camptothecintopoisomerase inhibitors have also been investigated as anticanceragents, including the indolocarbazole edotecarin, indenoisoquinolinesNSC 706744 (MJ-III-65), NSC 725776 (LMP-776), and NSC 724998 (LMP-400),dibenzonaphthyridiones such as topovale (ARC-111), and the aromathecinrosettacin (Pommier et al., DNA topoisomerases and their poisoning byanticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010)421-433).

In addition to the Top1 inhibitors, a number of anti-cancer agents havebeen investigated that target topoisomerase type II (Top2) enzymes,including etoposide, teniposide, and the DNA intercalators doxorubicin,daunorubicin, aclarubicin, amsacrine, dexrazoxane, TAS-103, thequinolone CP-115,963, the ellipticines including ellipticinium,azatoxins, genistein, VP-16, VM-26, mitoxantrone, amonafidem, andsaintopin.

One potential side-effect of the use of topoisomerase inhibitors asanti-cancer agents includes the development of secondary malignancies.For example, the use of etoposide induces treatment-related acutemyelocytic leukemia (t-AML) and treamtent related myelodysplasticsyndromes (t-MDS), which often progress to t-AML (Pedersen-Bjergaard etal., “Genetic pathways in therapy-related myelodysplasia and acutemyeloid leukemia,” Blood (2002) 99:1909-1912).

Previous studies have examined the potential cytotoxic activity ofcamptothecin derivatives in combination with CDK inhibitors againstsmall cell lung cancer cell lines. Hamilton et al. found that thenon-specific pan-CDK inhibitors olomoucine, roscovitine, and CDK4I had asynergistic cytotoxic effect on small cell lung cancer cell lines incombination with topotecan (“Synergistic Anticancer Activity ofTopotecan—Cyclin-Dependent Kinase Inhibitor Combinations againstDrug-Resistant Small Cell Lung Cancer (SCLC) Cell Lines”, Journal ofCancer Therapy (2013) 4: 47-53). In additional experiments, Hamilton etal. found that while the pan-CDK inhibitors olomoucine, roscovitine, andCDK4I had a synergistic cytotoxic effect on small cell lung cancer celllines in combination with various camptothecin derivatives (includingrubitecan, 9AC, topotecan, SN38, and CPT109), comparatively, the CDK 4/6inhibitor PD0332991 had low chemosensitizing activity (“Synergism ofCyclin-Dependent Kinase Inhibitors with Camptothecin Derivatives inSmall Cell Lung Cancer Cell Lines” Molecules (2014), 19(2): 2077-2088).

Accordingly, there is an ongoing need for improved compounds, methods,and regimes to treat patients with Rb-negative cancers and abnormalcellular proliferative disorders.

SUMMARY OF THE INVENTION

Methods and compositions are provided to treat abnormal cellularproliferation characterized by the loss, deficiency, or absence of theretinoblastoma tumor suppressor protein (Rb)(Rb-null or Rb-negative),including Rb-negative cancer, wherein a CDK4/6 inhibitor describedherein and a topoisomerase I inhibitor is administered in combination toa host resulting in an advantageous anti-tumor effect compared to theuse of either the CDK4/6 inhibitor, which does not inhibit Rb-negativecellular proliferation, or the topoisomerase I inhibitor alone.Suprisingly, this advantageous anti-tumor effect is seen at variousdosing levels of the CDK4/6 inhibitor, including low-dose levels of theCDK 4/6 inhibitor as described in the Examples below.

Furthermore, provided herein are methods and compositions for reducingthe development of secondary malignancies associated with the use oftopoisomerase inhibitors wherein a subject undergoing treatment for anRb-negative cancer with a topoisomerase inhibitor is administered aCDK4/6 inhibitor described herein.

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein and administered in acombination or alternation schedule with a topoisomerase I inhibitor. Inone non-limiting example, a CDK4/6 inhibitor compound selected fromTable 1 below is administered in combination or alternation with atopoisomerase I inhibitor. In one non-limiting example, a CDK4/6inhibitor selected from Table 1 below is administered in combination oralternation with a topoisomerase I inhibitor selected from the groupconsisting of(1S,9S)-1-Amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo(de)pyrano(3′,4′:6,7)indolizino(1,2-b)quinoline-10,13-dione(exatecan),(7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin(lurtotecan),(R)-5-ethyl-9,10-difluoro-5-hydroxy-4,5-dihydrooxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15(1H,13H)-dione(diflomotecan),(4S)-11-((E)-((1,1-Dimethylethoxy)imino)methyl)-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano(3′,4′:6,7)indolizino(1,2-b)quinoline-3,14(4H)-dione(gimatecan),(S)-8-ethyl-8-hydroxy-15-((4-methylpiperazin-1-yl)methyl)-11,14-dihydro-2H-[1,4]dioxino[2,3-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(3H,8H)-dione(lurtotecan),(4S)-4-Ethyl-4-hydroxy-11-[2-[(1-methylethyl)amino]ethyl]-1H-pyrano[3,4:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione(belotecan),6-((1,3-dihydroxypropan-2-yl)amino)-2,10-dihydroxy-12-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(edotecarin),8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxy-5H-dibenzo(c,h)(1,6)naphthyridin-6-one(topovale), benzo[6,7]indolizino[1,2-b]quinolin-11(13H)-one(rosettacin),(S)-4-ethyl-4-hydroxy-11-(2-(trimethylsilyl)ethyl)-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione(cositecan),tetrakis{(4S)-9-[([1,4′-bipiperidinyl]-1′-carbonyl)oxy]-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl}N,N′,N″,N′″-{methanetetrayltetrakis[methylenepoly(oxyethylene)oxy(1-oxoethylene)]}tetraglycinatetetrahydrochloride (etirinotecan pegol),(S)-10-amino-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione(9-aminocamptothecin), 10-hydroxy-camptothecin (HOCPT), or(S)-11-(tert-butyldimethylsilyl)-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione(silatecan),(R)-9-chloro-5-ethyl-5-hydroxy-10-methyl-12-((4-methylpiperidin-1-yl)methyl)-4,5-dihydrooxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15(1H,13H)-dione(elmotecan). In one embodiment, the CDK4/6 inhibitor is selected fromcompounds Q, T, U, and GG, or a combination thereof. In one embodiment,the CDK4/6 inhibitor is selected from compounds X and BB, or acombination thereof.

The subject treated according to the present invention may suffer from aproliferative disorder or disease such as cancer characterized by theloss, deficiency, or absence of the retinoblastoma (Rb) tumor suppressorprotein (Rb-null or Rb-negative). The cancer may be characterized byreduced expression of the retinoblastoma tumor suppressor protein or aretinoblastoma family member protein or proteins (such as, but notlimited to p107 and p130). Due to the lack or deficiency of Rb, thesecancers are generally CDK4/6 replication independent, and not growthinhibited upon exposure to a CDK4/6 inhibitor such as the CDK4/6inhibitors described herein.

In one embodiment, the subject is undergoing treatment for anRb-negative or Rb-deficient cancer, including but not limited to, smallcell lung cancer, triple-negative breast cancer, HPV-positive head andneck cancer, retinoblastoma, Rb-negative bladder cancer, Rb-negativeprostate cancer, osteosarcoma, or cervical cancer. In one embodiment,the cancer is selected from a gastric, glioma, non-small cell lung,esophageal, liver, breast, neuroblastoma, ovarian, sarcoma, for example,but not limited to Ewing sarcoma, pancreatic, lymphoma, prostate, orhematological cancer, for example, but not limited to acutelymphoblastic leukemia and acute myeloid leukemia, that has a loss,deficiency, or absence of the retinoblastoma (Rb) tumor suppressorprotein.

In one aspect of the invention, a subject is suffering from small celllung carcinoma and administered a CDK4/6 inhibitor selected from FormulaI, II, III, IV, or V as described herein, in a combination oralternation schedule with a topoisomerase I inhibitor. In oneembodiment, the topoisomerase I inhibitor is selected from exatecan,lurtotecan, diflomotecan, gimatecan, belotecan, edotecarin, topovale,rosettacin, cositecan, etirinotecan pegol, silatecan,9-aminocamptothecin, elmotecan, HOCPT, or a combination thereof. In oneembodiment of the invention, a subject suffering from small cell lungcarcinoma is administered a CDK4/6 inhibitor selected from Formula I,II, III, IV, or V, in combination or alternation with exatecan,lurtotecan, diflomotecan, gimatecan, belotecan, edotecarin, topovale,rosettacin, cositecan, etirinotecan pegol, silatecan,9-aminocamptothecin, HOCPT, or a combination thereof. In anotherembodiment of the invention, a subject suffering from small cell lungcarcinoma is administered a CDK4/6 inhibitor selected from compounds Q,T, U, GG, X, and BB, or a combination thereof, in combination oralternation with exatecan, lurtotecan, diflomotecan, gimatecan,belotecan, edotecarin, topovale, rosettacin, cositecan, etirinotecanpegol, silatecan, 9-aminocamptothecin, elmotecan, HOCPT, or acombination thereof. In one embodiment of the invention, a subjectsuffering from small cell lung carcinoma is administered a CDK4/6inhibitor selected from compounds Q, T, U, GG, X, and BB, or acombination thereof, in combination or alternation with exatecan. In oneembodiment of the invention, a subject suffering from small cell lungcarcinoma is administered a CDK4/6 inhibitor selected from compounds Q,T, U, GG, X, and BB, or a combination thereof, in combination oralternation with lurtotecan. In one embodiment of the invention, asubject suffering from small cell lung carcinoma is administered aCDK4/6 inhibitor selected from compounds Q, T, U, GG, X, and BB, or acombination thereof, in combination or alternation with diflomotecan. Ina further embodiment of the invention, a subject suffering from smallcell lung carcinoma is administered a CDK4/6 inhibitor selected fromcompounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with belotecan. In one embodiment of theinvention, a subject suffering from small cell lung carcinoma isadministered a CDK4/6 inhibitor selected from compounds Q, T, U, GG, X,and BB, or a combination thereof, in combination or alternation withedotecarin. In one embodiment of the invention, a subject suffering fromsmall cell lung carcinoma is administered a CDK4/6 inhibitor selectedfrom compounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with topovale. In one embodiment of theinvention, a subject suffering from small cell lung carcinoma isadministered a CDK4/6 inhibitor selected from compounds Q, T, U, GG, X,and BB, or a combination thereof, in combination or alternation withrosettacin. In one embodiment of the invention, a subject suffering fromsmall cell lung carcinoma is administered a CDK4/6 inhibitor selectedfrom compounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with cositecan. In one embodiment of theinvention, a subject suffering from small cell lung carcinoma isadministered a CDK4/6 inhibitor selected from compounds Q, T, U, GG, X,and BB, or a combination thereof, in combination or alternation withetirinotecan pegol. In yet another embodiment of the invention, asubject suffering from small cell lung carcinoma is administered aCDK4/6 inhibitor selected from compounds Q, T, U, GG, X, and BB, or acombination thereof, in combination or alternation with silatecan. Inone embodiment of the invention, a subject suffering from small celllung carcinoma is administered a CDK4/6 inhibitor selected fromcompounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with 9-aminocamptothecin. In one embodimentof the invention, a subject suffering from small cell lung carcinoma isadministered a CDK4/6 inhibitor selected from compounds Q, T, U, GG, X,and BB, or a combination thereof, in combination or alternation withelmotecan. In one embodiment of the invention, a subject suffering fromsmall cell lung carcinoma is administered a CDK4/6 inhibitor selectedfrom compounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with HOCPT.

In another aspect of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with a topoisomerase I inhibitor toa subject having an Rb-negative cervical cancer. In one embodiment, thetopoisomerase I inhibitor is selected from topotecan, exatecan,lurtotecan, diflomotecan, gimatecan, belotecan, edotecarin, topovale,rosettacin, cositecan, etirinotecan pegol, 9-aminocamptothecin,silatecan, elmotecan, HOCPT, or a combination thereof. In one embodimentof the invention, a subject suffering from Rb-negative cervical cancerand administered a CDK4/6 inhibitor selected from compounds Q, T, U, GG,X, and BB, or a combination thereof, in combination or alternation withtopotecan, exatecan, lurtotecan, diflomotecan, gimatecan, belotecan,edotecarin, topovale, rosettacin, cositecan, etirinotecan pegol,9-aminocamptothecin, silatecan, elmotecan, HOCPT, or a combinationthereof. In an additional embodiment of the invention, a subjectsuffering from Rb-negative cervical cancer and administered a CDK4/6inhibitor selected from compounds Q, T, U, GG, X, and BB, or acombination thereof, in combination or alternation with topotecan.

In one aspect of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with a topoisomerase I inhibitor toa subject having an Rb-negative ovarian cancer. In one embodiment, thetopoisomerase I inhibitor is selected from topotecan, exatecan,lurtotecan, diflomotecan, gimatecan, belotecan, edotecarin, topovale,rosettacin, cositecan, etirinotecan pegol, 9-aminocamptothecin,silatecan, elmotecan, HOCPT, or a combination thereof. In anotherembodiment of the invention, a subject suffering from Rb-negativeovarian cancer and administered a CDK4/6 inhibitor selected fromcompounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with topotecan, exatecan, lurtotecan,diflomotecan, gimatecan, belotecan, edotecarin, topovale, rosettacin,cositecan, etirinotecan pegol, 9-aminocamptothecin, silatecan,elmotecan, HOCPT, or a combination thereof. In a further embodiment ofthe invention, a subject suffering from Rb-negative ovarian cancer andadministered a CDK4/6 inhibitor selected from compounds Q, T, U, GG, X,and BB, or a combination thereof, in combination or alternation withtopotecan.

In a further aspect of the invention, a CDK4/6 inhibitor is selectedfrom Formula I, II, III, IV, or V as described herein, and administeredin a combination or alternation schedule with a topoisomerase Iinhibitor to a subject having an Rb-negative neuroblastoma. In oneembodiment, the topoisomerase I inhibitor is selected from topotecan,exatecan, lurtotecan, diflomotecan, gimatecan, belotecan, edotecarin,topovale, rosettacin, cositecan, etirinotecan pegol,9-aminocamptothecin, silatecan, elmotecan, HOCPT, or a combinationthereof. In another embodiment of the invention, a subject sufferingfrom Rb-negative neuroblastoma and administered a CDK4/6 inhibitorselected from compounds Q, T, U, GG, X, and BB, or a combinationthereof, in combination or alternation with topotecan, exatecan,lurtotecan, diflomotecan, gimatecan, belotecan, edotecarin, topovale,rosettacin, cositecan, etirinotecan pegol, 9-aminocamptothecin,silatecan, elmotecan, HOCPT, or a combination thereof. In an additionalembodiment of the invention, a subject suffering from Rb-negativeneuroblastoma and administered a CDK4/6 inhibitor selected fromcompounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with topotecan.

In one aspect of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with a topoisomerase I inhibitor toa subject having an Rb-negative sarcoma, for example, Ewing sarcoma. Inone embodiment, the topoisomerase I inhibitor is selected fromtopotecan, exatecan, lurtotecan, diflomotecan, gimatecan, belotecan,edotecarin, topovale, rosettacin, cositecan, etirinotecan pegol,9-aminocamptothecin, silatecan, elmotecan, HOCPT, or a combinationthereof. In another embodiment of the invention, a subject sufferingfrom Rb-negative sarcoma and administered a CDK4/6 inhibitor selectedfrom compounds Q, T, U, GG, X, and BB, or a combination thereof, incombination or alternation with topotecan, exatecan, lurtotecan,diflomotecan, gimatecan, belotecan, edotecarin, topovale, rosettacin,cositecan, etirinotecan pegol, 9-aminocamptothecin, silatecan,elmotecan, HOCPT, or a combination thereof. In an additional embodimentof the invention, a subject suffering from Rb-negative sarcoma andadministered a CDK4/6 inhibitor selected from compounds Q, T, U, GG, X,and BB, or a combination thereof, in combination or alternation withtopotecan.

In another aspect of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with a topoisomerase I inhibitor toa subject having an Rb-negative leukemia, for example, acute myeloidleukemia (AML) or acute lympoblastic leulkemia (ALL). In one embodiment,the topoisomerase I inhibitor is selected from topotecan, exatecan,lurtotecan, diflomotecan, gimatecan, belotecan, edotecarin, topovale,rosettacin, cositecan, etirinotecan pegol, 9-aminocamptothecin,silatecan, elmotecan, HOCPT, or a combination thereof. In anotherembodiment of the invention, a subject suffering from Rb-negativeleukemia and administered a CDK4/6 inhibitor selected from compounds Q,T, U, GG, X, and BB, or a combination thereof, in combination oralternation with topotecan, exatecan, lurtotecan, diflomotecan,gimatecan, belotecan, edotecarin, topovale, rosettacin, cositecan,etirinotecan pegol, 9-aminocamptothecin, silatecan, elmotecan, HOCPT, ora combination thereof. In a further embodiment of the invention, asubject suffering from Rb-negative leukemia and administered a CDK4/6inhibitor selected from compounds Q, T, U, GG, X, and BB, or acombination thereof, in combination or alternation with topotecan.

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with diflomotecan to a subjecthaving an Rb-negative breast, small cell lung, or prostate cancer.

In another embodiment of the invention, a CDK4/6 inhibitor is selectedfrom Formula I, II, III, IV, or V as described herein, and administeredin a combination or alternation schedule with gimatecan to a subjecthaving an Rb-negative glioblastoma or small cell lung cancer.

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with lurtotecan to a subject havingan Rb-negative small cell lung cancer or ovarian cancer.

In a further embodiment of the invention, a CDK4/6 inhibitor is selectedfrom Formula I, II, III, IV, or V as described herein, and administeredin a combination or alternation schedule with exatecan to a subjecthaving an Rb-negative sarcoma, for example Ewing's sarcoma, pancreatic,gastric, or liver cancer.

In an additional embodiment of the invention, a CDK4/6 inhibitor isselected from Formula I, II, III, IV, or V as described herein, andadministered in a combination or alternation schedule with belotecan toa subject having an Rb-negative small cell lung, cervical, non-smallcell lung, or ovarian cancer.

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with cositecan to a subject havingan Rb-negative cervical, non-small cell lung, glioblastoma, melanoma, orovarian cancer.

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with etirinotecan pegol to a subjecthaving an Rb-negative non-small cell lung, small cell lung,glioblastoma, or breast cancer.

In another embodiment of the invention, a CDK4/6 inhibitor is selectedfrom Formula I, II, III, IV, or V as described herein, and administeredin a combination or alternation schedule with 9-aminocamptothecin to asubject having an Rb-negative leukemia, ovarian, esophageal, gastric,lymphoma, or small cell lung cancer.

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with silatecan to a subject havingan Rb-negative glioblastoma.

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with topotecan to a subject havingan Rb-negative small cell lung cancer. In one embodiment, the CDK 4/6inhibitor is selected from compounds Q, T, U, GG, X, and BB.

In one aspect of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, and administered in acombination or alternation schedule with a topoisomerase II inhibitor toa subject suffering from an Rb-negative cancer. In one non-limitingexample, a CDK4/6 inhibitor compound selected from Table 1 below isadministered in combination or alternation with a topoisomerase IIinhibitor. In one non-limiting example, a CDK4/6 inhibitor selected fromTable 1 below is administered in combination or alternation with atopoisomerase II inhibitor selected from etoposide, teniposide, and theDNA intercalators doxorubicin, daunorubicin, aclarubicin, amsacrine,dexrazoxane, TAS-103, the quinolone CP-115,963, the ellipticinesincluding ellipticinium, azatoxins, genistein, VP-16, VM-26,mitoxantrone, amonafidem, or saintopin.

In one embodiment of the invention, the combination therapy describedherein can be administered in a concerted regimen with at least oneother chemotherapeutic agent, targeted anti-neoplastic agent,immunotherapeutic agent, or a hematopoietic growth factor agent forbeneficial, additive, or further synergistic effects against theabnormal cellular proliferation.

In certain embodiments, a CDK4/6 inhibitor described herein isadministered to the subject prior to treatment with the topoisomeraseinhibitor, during treatment with the topoisomerase inhibitor, afteradministration of the topoisomerase inhibitor, or a combination thereof.In one embodiment, a CDK4/6 inhibitor described herein is administeredto the subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8hours, 4 hours, 2 hours, 1 hour, 30 minutes or less prior to treatmentwith the topoisomerase inhibitor. In one embodiment, the CDK4/6inhibitor is administered up to 4 hours prior to treatment with thetopoisomerase inhibitor. In one embodiment, the CDK4/6 inhibitor isadministered approximately 30 minutes prior to treatment with thetopoisomerase inhibitor. In one embodiment, the CDK4/6 inhibitor isadministered at approximately the same time as the topoisomeraseinhibitor. In one embodiment, the CDK4/6 inhibitor is administeredwithin 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16hours, 20 hours, or 24 hours after treatment with the topoisomeraseinhibitor.

In summary, the present invention includes the following features:

A) Methods for treating a host suffering from an Rb-negativeproliferative disorder, for example but not limited to an Rb-negativecancer, comprising administering an effective amount of a compound ofFormula I, II, III, IV, or V, or a salt, isotopic analog, or prodrugthereof, including a compound selected from Table 1 as described herein,in combination with a topoisomerase inhibitor;B) A pharmaceutically acceptable composition for use as achemotherapeutic comprising a CDK4/6 inhibitor described herein, or asalt, isotopic analog, or prodrug thereof, and a topoisomeraseinhibitor;C) Use of a CDK4/6 inhibitor described herein, or a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof, incombination with a topoisomerase inhibitor, in the manufacture of amedicament for use as a chemotherapeutic to treat a subject with anRb-negative abnormal cellular proliferation disorder, including anRb-negative cancer;D) Processes for the preparation of therapeutic products that contain aneffective amount of a CDK 4/6 inhibitor compound described herein incombination with a topoisomerase inhibitor, for use in the treatment ofa subject having an Rb-negative abnormal cellular proliferationdisorder, such as an Rb-negative cancer, and;E) A method for manufacturing a medicament selected from a CDK4/6inhibitor described herein in combination with a topoisomeraseinhibitor, intended for therapeutic use as a chemotherapeutic for thetreatment of an Rb-negative abnormal cellular proliferation disorder,such as an Rb-negative cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 illustrate several exemplary embodiments of R² of thecompounds of CDK4/6 inhibitors useful in the present invention.

FIGS. 4A-4C, 5A-5D, 6A-6C, 7A-7B, and 8A-8F illustrate several exemplaryembodiments of the core structure CDK4/6 inhibitors useful in thepresent invention of the compounds of the invention.

FIG. 9A is a graph of the percentage of cells in G2-M phase (opencircles), S phase (triangles), G0-G1 phase (squares), <2N (diamonds) vs.variable concentration (nM) of compound T in tHS68 cells. TheRb-positive cell line (tHS68) was treated with the indicatedconcentrations of Compound T for 24 hours. Following treatment ofCompound T, cells were harvested and analyzed for cell cycledistribution. As described in Example 153, tHS68 cells show a clean G1arrest accompanied by a corresponding decrease in the number of cells inS-phase.

FIG. 9B is a graph of the number of tHS68 cells (Rb-positive cell line)vs. the DNA content of the cells (as measured by propidium iodide).Cells were treated with DMSO for 24 hours, harvested, and analyzed forcell cycle distribution.

FIG. 9C is a graph of the number of WM2664 cells (Rb-positive cell line)vs. the DNA content of the cells (as measured by propidium iodide).Cells were treated with DMSO for 24 hours, harvested, and analyzed forcell cycle distribution.

FIG. 9D is a graph of the number of A2058 cells (Rb-negative cell line)vs. the DNA content of the cells (as measured by propidium iodide).Cells were treated with DMSO for 24 hours, harvested, and analyzed forcell cycle distribution.

FIG. 9E is a graph of the number of tHS68 cells (Rb-positive cell line)vs. the DNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 161, treatment of tHS68 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow).

FIG. 9F is a graph of the number of WM2664 cells (Rb-positive cell line)vs. the DNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 161, treatment of WM2664 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow).

FIG. 9G is a graph of the number of A2058 cells (Rb-negative cell line)vs. the DNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 154, treatment of A2058 cells with Compound T doesnot cause a loss of the S-phase peak (indicated by arrow).

FIG. 10 is a Western blot showing the phosphorylation levels of Rb atSer807/811 and Ser780 after treatment with Compound T. Rb-positive(tHS68 or WM2664) and Rb-negative cell lines (A2058) were treated withCompound T (300 nM) for the indicated times (0, 4, 8, 16, and 24 hours).MAPK levels are shown as a control for protein levels. Followingtreatment, cells were harvested and analyzed for Rb-phosphorylation bywestern blot analysis. As described in Example 155, Compound T treatmentresulted in reduced Rb-phosphorylation starting 16 hours after treatmentin Rb-positive cell lines (tHS68 and WM2664), but not in the Rb-negativecell line (A2058).

FIG. 11 is a graph showing the tumor size (in cubic millimeters) in aCDK4/6 independent mouse model (NCI-H69 SCLC xenograft model) treatedwith vehicle control (closed squares), Compound T (open squares),carboplatin/etoposide (closed triangles), CompoundT/carboplatin/etoposide (open triangles), topotecan (closed circles), orCompound T/topotecan (open circles). As discussed in Example 156,Compound T showed enhanced efficacy in slowing tumor growth incombination with the chemotherapeutic agent topotecan. In contrast,Compound T did not result in increased efficacy in slowing tumor growthin combination with the chemotherapeutic agents carboplatin andetoposide.

FIG. 12 is a graph showing the tumor size (log₁₀ mean tumor volume) vs.time (days) in a CDK4/6 independent tumor mouse model (NCI-H69 SCLCxenograft model) treated with Compound T vehicle and topotecan vehiclecontrol (closed squares). As discussed in Example 157, the tumordoubling time for the NCI-H69 SCLC xenograft model was 5.8 days.

FIG. 13 is a graph showing animal weight (grams) vs. time (days) in aCDK4/6 independent tumor mouse model (NCI-H69 SCLC xenograft model).Tumor-bearing mice were treated with Compound T vehicle and topotecanvehicle (Group 1; closed squares), Compound T (100 mg/kg) and topotecanvehicle (Group 2; open squares), Compound T (100 mg/kg) and topotecan(0.6 mg/kg) (Group 3; closed triangles), Compound T (50 mg/kg) andtopotecan (0.6 mg/kg) (Group 4; open triangles), Compound T (10 mg/kg)and topotecan (0.6 mg/kg) (Group 5; closed circles) or Compound Tvehicle and topotecan (0.6 mg/kg) (Group 6; open circles). As discussedin Example 157, Compound T, administered alone or thirty minutes beforetopotecan, was well tolerated with no additive weight loss or toxicity.

FIG. 14 is a graph showing animal weight change (%) vs. time (days) in aCDK4/6 independent tumor mouse model (NCI-H69 SCLC xenograft model).Tumor-bearing mice were treated with Compound T vehicle and topotecanvehicle (Group 1; closed squares), Compound T (100 mg/kg) and topotecanvehicle (Group 2; open squares), Compound T (100 mg/kg) and topotecan(0.6 mg/kg) (Group 3; closed triangles), Compound T (50 mg/kg) andtopotecan (0.6 mg/kg) (Group 4; open triangles), Compound T (10 mg/kg)and topotecan (0.6 mg/kg) (Group 5; closed circles) or Compound Tvehicle and topotecan (0.6 mg/kg) (Group 6; open circles). As discussedin Example 157, the percent weight gain versus Day 0 in each group ofanimals was similar.

FIG. 15 is a graph showing survival (% subjects remaining) vs. time(days) in a CDK4/6 independent mouse model (NCI-H69 SCLC xenograftmodel). Tumor-bearing mice were treated with Compound T vehicle andtopotecan vehicle (Group 1), Compound T (100 mg/kg) and topotecanvehicle (Group 2), Compound T (100 mg/kg) and topotecan (0.6 mg/kg)(Group 3), Compound T (50 mg/kg) and topotecan (0.6 mg/kg) (Group 4),Compound T (10 mg/kg) and topotecan (0.6 mg/kg) (Group 5) or Compound Tvehicle and topotecan (0.6 mg/kg) (Group 6). As discussed in Example157, mice treated with topotecan or topotecan plus Compound T survivedsignificantly longer than mice treated with vehicle or Compound T only.The combination of topotecan with 100 mg/kg Compound T or 50 mg/kgCompound T led to increased survival rates in comparison to topotecanalone.

FIG. 16 is a graph showing tumor volume (mm³) vs. time (days) in aCDK4/6 independent mouse model (NCI-H69 SCLC xenograft model).Tumor-bearing mice were treated with Compound T vehicle and topotecanvehicle (Group 1; closed squares), Compound T (100 mg/kg) and topotecanvehicle (Group 2; open squares), Compound T (100 mg/kg) and topotecan(0.6 mg/kg) (Group 3; closed triangles), Compound T (50 mg/kg) andtopotecan (0.6 mg/kg) (Group 4; open triangles), Compound T (10 mg/kg)and topotecan (0.6 mg/kg) (Group 5; closed circles) or Compound Tvehicle and topotecan (0.6 mg/kg) (Group 6; open circles). As discussedin Example 157, Compound T alone was inactive toward NCI-H69 xenografts,while topotecan alone was active and reduced tumor volumes (p<0.0001).Additionally, the combination of topotecan with each concentration ofCompound T tested (100 mg/kg, 50 mg/kg, or 10 mg/kg) led to a reductionin tumor volume in comparison to treatment with vehicle alone(p<0.0001).

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions are provided to treat abnormal cellularproliferation characterized by the loss, deficiency, or absence of theretinoblastoma tumor suppressor protein (Rb)(Rb-null or Rb-negative),including Rb-negative cancer, wherein a CDK4/6 inhibitor describedherein and a topoisomerase inhibitor, for example a topoisomerase Iinhibitor, is administered in combination or alternation to a subjectresulting in an advantageous effect compared to the use of either theCDK4/6 inhibitor or topoisomerase inhibitor alone. In a typicalembodiment, the subject is a human.

Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Definition of standard chemistryterms may be found in reference works, including Carey and Sundberg(2007) Advanced Organic Chemistry 5^(th) Ed. Vols. A and B, SpringerScience+Business Media LLC, New York. The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof synthetic organic chemistry, mass spectroscopy, preparative andanalytical methods of chromatography, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology. Conventional methods oforganic chemistry include those included in March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, 6^(th) Edition, M. B.Smith and J. March, John Wiley & Sons, Inc., Hoboken, N.J., 2007.

The term “alkyl,” either alone or within other terms such as “haloalkyl”and “alkylamino,” embraces linear or branched radicals having one toabout twelve carbon atoms. “Lower alkyl” radicals have one to about sixcarbon atoms. Examples of such radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl and the like. The term “alkylene” embraces bridging divalentlinear and branched alkyl radicals. Examples include methylene,ethylene, propylene, isopropylene and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twelve carbon atoms.“Lower alkenyl” radicals having two to about six carbon atoms. Examplesof alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl” and “lower alkenyl,” embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” denotes linear or branched radicals having at leastone carbon-carbon triple bond and having two to about twelve carbonatoms. “Lower alkynyl” radicals having two to about six carbon atoms.Examples of such radicals include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl radicals may be optionally substituted withone or more functional groups such as halo, hydroxy, nitro, amino,cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “alkylamino” embraces “N-alkylamino” and “N,N-dialkylamino”where amino groups are independently substituted with one alkyl radicaland with two alkyl radicals, respectively. “Lower alkylamino” radicalshave one or two alkyl radicals of one to six carbon atoms attached to anitrogen atom. Suitable alkylamino radicals may be mono or dialkylaminosuch as N-methylamino, N-ethylamino, N.N-dimethylamino, N,N-diethylaminoand the like.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “haloalkyl” embraces radicals wherein any one or more of thealkyl carbon atoms is substituted with one or more halo as definedabove. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkylradicals including perhaloalkyl. A monohaloalkyl radical, for oneexample, may have an iodo, bromo, chloro or fluoro atom within theradical. Dihalo and polyhaloalkyl radicals may have two or more of thesame halo atoms or a combination of different halo radicals. “Lowerhaloalkyl” embraces radicals having 1-6 carbon atoms. Examples ofhaloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Perfluoroalkyl” means an alkyl radical having allhydrogen atoms replaced with fluoro atoms. Examples includetrifluoromethyl and pentafluoroethyl.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one or two rings wherein such rings may be attachedtogether in a fused manner. The term “aryl” embraces aromatic radicalssuch as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. Morepreferred aryl is phenyl. Said “aryl” group may have 1 or moresubstituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro,cyano, alkoxy, lower alkylamino, and the like. An aryl group may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “heterocyclyl” (or “heterocyclo”) embraces saturated, andpartially saturated heteroatom-containing ring radicals, where theheteroatoms may be selected from nitrogen, sulfur and oxygen. In oneembodiment, heterocyclic rings comprise monocyclic 6-8 membered rings,as well as 5-16 membered bicyclic ring systems (which can includebridged fused and spiro-fused bicyclic ring systems). It does notinclude rings containing —O—O—.—O—S— or —S—S— portions. Said“heterocyclyl” group may have one or more substituents such as hydroxyl,Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, loweralkoxy, amino, lower alkylamino, and the like. In one embodiment, said“heterocyclyl” group may have 1 to 3 substituents such as hydroxyl, Boc,halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy,amino, lower alkylamino, and the like. In an alternate embodiment, aheterocyclic ring comprises a monocyclic 3-6 membered ring.

Examples of saturated heterocyclo groups include saturated 3- to6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms[e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,piperazinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,thiazolidinyl]. Examples of partially saturated heterocyclyl radicalsinclude dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl,and the like.

Particular examples of partially saturated and saturated heterocyclogroups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl,thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl,indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl,isochromanyl, chromanyl, 1,2-dihydroquinolyl,1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl,2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl,5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl,3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl,2,3-dihydro-1H-1λ′-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryland dihydrothiazolyl, and the like.

Heterocyclo groups also includes radicals where heterocyclic radicalsare fused/condensed with aryl radicals: unsaturated condensedheterocyclic group containing 1 to 5 nitrogen atoms, for example,indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo[1,5-b]pyridazinyl]; unsaturated condensed heterocyclic group containing1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. benzoxazolyl,benzoxadiazolyl]; unsaturated condensed heterocyclic group containing 1to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., benzothiazolyl,benzothiadiazolyl]; and saturated, partially unsaturated and unsaturatedcondensed heterocyclic group containing 1 to 2 oxygen or sulfur atoms[e.g. benzofuryl, benzothienyl, 2,3-dihydro-benzo[1,4]dioxinyl anddihydrobenzofuryl].

The term “heteroaryl” denotes aryl ring systems that contain one or moreheteroatoms selected from the group O, N and S, wherein the ringnitrogen and sulfur atom(s) are optionally oxidized, and nitrogenatom(s) are optionally quarternized. Examples include unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g.,4H-1,2,4-triazolyl, IH-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated5- to 6-membered heteromonocyclic group containing an oxygen atom, forexample, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-memberedheteromonocyclic group containing a sulfur atom, for example, 2-thienyl,3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example,oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g.,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl]. Said“heteroaryl” group may have one or more substituents such as hydroxyl,Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, loweralkoxy, amino, lower alkylamino, and the like.

The term “heteroarylalkyl” denotes alkyl radicals substituted with aheteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term “sulfonyl”, whether used alone or linked to other terms such asalkylsulfonyl, denotes respectively divalent radicals —SO₂—.

The terms “carboxy” or “carboxyl”, whether used alone or with otherterms, such as “carboxyalkyl”, denotes —C(O)—OH.

The term “carbonyl”, whether used alone or with other terms, such as“aminocarbonyl”, denotes —C(O)—.

The term “aminocarbonyl” denotes an amide group of the Formula—C(O)—NH₂.

The terms “heterocycloalkyl” embrace heterocyclic-substituted alkylradicals. Examples include piperidylmethyl and morpholinylethyl.

The term “arylalkyl” embraces aryl-substituted alkyl radicals. Examplesinclude benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, halkoalkyl andhaloalkoxy.

The term “cycloalkyl” includes saturated carbocyclic groups of 3 to 10carbons. Lower cycloalkyl groups include C₃-C₆ rings. Examples includecyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “cycloalkylalkyl” embraces cycloalkyl-substituted alkylradicals. “Lower cycloalkylalkyl” radicals are cycloalkyl radicalsattached to alkyl radicals having one to six carbon atoms. Examples ofinclude cyclohexylmethyl. The cycloalkyl in said radicals may beadditionally substituted with halo, alkyl, alkoxy and hydroxy.

The term “cycloalkenyl” includes carbocyclic groups having one or morecarbon-carbon double bonds including “cycloalkyldienyl” compounds.Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl andcycloheptadienyl.

The term “comprising” is meant to be open ended, including the indicatedcomponent but not excluding other elements.

The term “oxo” as used herein contemplates an oxygen atom attached witha double bond.

The term “nitro” as used herein contemplates —NO₂.

The term “cyano” as used herein contemplates —CN.

As used herein, the term “prodrug” means a compound which whenadministered to a host in vivo is converted into the parent drug. Asused herein, the term “parent drug” means any of the presently describedchemical compounds that are useful to treat any of the disordersdescribed herein, or to control or improve the underlying cause orsymptoms associated with any physiological or pathological disorderdescribed herein in a host, typically a human. Prodrugs can be used toachieve any desired effect, including to enhance properties of theparent drug or to improve the pharmaceutic or pharmacokinetic propertiesof the parent. Prodrug strategies exist which provide choices inmodulating the conditions for in vivo generation of the parent drug, allof which are deemed included herein. Nonlimiting examples of prodrugstrategies include covalent attachment of removable groups, or removableportions of groups, for example, but not limited to acylation,phosphorylation, phosphonylation, phosphoramidate derivatives,amidation, reduction, oxidation, esterification, alkylation, othercarboxy derivatives, sulfoxy or sulfone derivatives, carbonylation oranhydride, among others.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist, unless otherwise noted.

The term “selective CDK4/6 inhibitor” used in the context of thecompounds described herein includes compounds that inhibit CDK4activity, CDK6 activity, or both CDK4 and CDK6 activity at an IC₅₀ molarconcentration at least about 500 times less than the IC₅₀ molarconcentration necessary to inhibit to the same degree of CDK2 activityin a standard phosphorylation assay. In alternative embodiments, theIC₅₀ molar concentration is at least about 1000, or 1500, 1800 or 2000times less than the IC₅₀ molar concentration necessary to inhibit to thesame degree of CDK2 activity in a standard phosphorylation assay.

As used herein the term “chemotherapy” or “chemotherapeutic agent”refers to treatment with a cytostatic or cytotoxic agent (i.e., acompound) to reduce or eliminate the growth or proliferation ofundesirable cells, for example cancer cells. Thus, as used herein,“chemotherapy” or “chemotherapeutic agent” refers to a cytotoxic orcytostatic agent used to treat a proliferative disorder, for examplecancer.

The host is typically a human, although it is to be understood themethods described herein are effective for other animals, such asmammals and vertebrate species. More particularly, the term host caninclude animals either for veterinary purposes or used in assays such asthose used in preclinical testing including but not limited to mice,rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine(pigs and hogs), ruminants, equine, poultry, felines, bovines, murines,canines, and the like.

Active Compounds

In one embodiment, the invention is directed to methods of administeringa CDK4/6 inhibitor in combination or alternation with a topoisomerase Iinhibitor to treat an Rb-negative cellular proliferation disorder, suchas cancer, wherein the CDK4/6 inhibitor is selected from Formula I, II,III, IV, or V:

or a pharmaceutically acceptable salt thereof;wherein:Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is2, 3 or 4;

each X is independently CH or N;

each X′ is independently CH or N;

X″ is independently CH₂, S or NH, arranged such that the moiety is astable 5-membered ring; R, R⁸, and R¹¹ are independently H, C₁-C₃ alkylor haloalkyl, cycloalkyl or cycloalkyl containing one or moreheteroatoms selected from N, O or S; -(alkylene)m-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)n-NR³R⁴ any of whichmay be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring;

each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, whereineach of said alkyl, cycloalkyl and haloalkyl groups optionally includesO or N heteroatoms in place of a carbon in the chain and two R¹'s onadjacent ring atoms or on the same ring atom together with the ringatom(s) to which they are attached optionally form a 3-8-membered cycle;y is 0, 1, 2, 3 or 4;R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2;R³ and R⁴ at each occurrence are independently:

-   -   (i) hydrogen or    -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,        cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring; or R³ and R⁴ together with the nitrogen        atom to which they are attached may combine to form a        heterocyclo ring optionally independently substituted with one        or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring;        R⁵ and R⁵* at each occurrence is:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valance;        R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(O)—OR⁵) -(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:    -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups        may be further independently substituted with one or more        -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵,        -(alkylene)_(m)-S(O)_(n-)R⁵*, -(alkylene)_(m)-NR³*R⁴*,        -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,        -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,        -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,        -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,    -   n is 0, 1 or 2, and    -   m is 0 or 1;        R³* and R⁴* at each occurrence are independently:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valance;        or R³* and R⁴* together with the nitrogen atom to which they are        attached may combine to form a heterocyclo ring optionally        independently substituted with one or more R^(x) groups as        allowed by valance; and        R⁶ is H or lower alkyl, -(alkylene)_(m)-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atoms may optionally        combine to form a ring; and        R¹⁰ is (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        or a pharmaceutically acceptable salt, prodrug or isotopic        variant, for example, partially or fully deuterated form        thereof.

In some aspects, the CDK4/6 inhibitor used in the combination is FormulaI or Formula II and R⁶ is absent.

In some aspects, the CDK4/6 inhibitor used in the combination is ofFormula III:

and the variables are as defined for compounds of Formulae I and II andor a pharmaceutically acceptable salt, prodrug, or isotopic variant, forexample, partially or fully deuterated form thereof.

In some aspects, R^(x) is not further substituted.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸ is hydrogen or C₁-C₃ alkyl.

In some aspects, R is hydrogen or C₁-C₃ alkyl.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ any of which may beoptionally independently substituted with one or more R^(x) groups asallowed by valance, and wherein two R^(x) groups bound to the same oradjacent atom may optionally combine to form a ring.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ without furthersubstitution.

In some aspects, m in R² is 1. In a further aspect, the alkylene in R²is methylene.

In some aspects, R² is

wherein:R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)- or-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;each R^(x1) is independently-(alkylene)_(m)-(C(O))_(m)-(alkylene)_(m)-(N(R^(N)))_(m)-(alkyl)_(m)wherein each m is independently 0 or 1 provided at least one m is 1,—(C(O))—O-alkyl, -(alkylene)_(m)-cycloalkyl wherein m is 0 or 1,—N(R^(N))-cycloalkyl, —C(O)-cycloalkyl, -(alkylene)_(m)-heterocyclylwherein m is 0 or 1, or —N(R^(N))-heterocyclyl, —C(O)-heterocyclyl,—S(O)₂-(alkylene)_(m) wherein m is 1 or 2, wherein:

-   -   R^(N) is H, C₁ to C₄ alkyl or C₁ to C₆ heteroalkyl, and    -   wherein two R^(x1) can, together with the atoms to which they        attach on P, which may be the same atom, form a ring; and        t is 0, 1 or 2.

In some aspects, each R^(x1) is only optionally substituted byunsubstituted alkyl, halogen or hydroxy.

In some aspects, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl.

In some aspects, at least one R^(x1) is -(alkylene)_(m)-heterocyclylwherein m is 0 or 1.

In some aspects, R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some aspects, R² is

In some aspects, R² is

In some aspects, R² is

wherein:R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)- or-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;P1 is a 4- to 6-membered monocyclic saturated heterocyclyl group;each R^(x2) is independently hydrogen or alkyl; ands is 0, 1 or 2.

In some aspects, R² is

In some aspects, P1 includes at least one nitrogen.

In some aspects, any alkylene in R²* in any previous aspect is notfurther substituted.

In some aspects, R² is selected from the structures depicted in FIGS.1-3.

In some aspects, R² is

In some aspects, the CDK4/6 inhibitor used has general Formula I andmore specifically one of the general structures in FIGS. 4-8 wherein thevariables are as previously defined.

In some aspects, the CDK4/6 inhibitor used has general Formula Ia:

wherein R¹, R², R and y are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl andR^(2*) is as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib:

wherein R² and R are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic:

wherein R² and R are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Id:

wherein R² and R are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ie:

In some embodiments, the CDK4/6 inhibitor used has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula If:

In some embodiments, the CDK4/6 inhibitor used has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Ig:

In some embodiments, the CDK4/6 inhibitor used has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ih:

In some embodiments, the CDK4/6 inhibitor used has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ii:

In some embodiments, the CDK4/6 inhibitor used has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij:

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, R is hydrogen in Formula Ia, Formula Ib, FormulaIc, Formula Id, Formula Ie, Formula If, Formula Ig, Formula Ih, FormulaIi, and Formula Ij.

In some embodiments, R is alkyl in Formula Ia, Formula Ib, Formula Ic,Formula Id, Formula Ie, Formula If, Formula Ig, Formula Ih, Formula Ii,and Formula Ij.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R isH, and both X are N.

In some embodiments, the CDK4/6 inhibitor used has the structure:

In some embodiments, the CDK4/6 inhibitor used has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Il:

In some embodiments, the CDK4/6 inhibitor used has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments the CDK4/6 inhibitor used has Formula Im:

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula IIa:

In some embodiments, the CDK4/6 inhibitor used has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula IIb:

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some aspects, the CDK4/6 inhibitor used is:

Further specific CDK4/6 inhibitor compounds that can be used incombination with topoisomerase inhibitors and can be used in thedisclosed methods of treatment and compositions include the structureslisted in Table 1 below.

TABLE 1 Nonlimiting Examples of Compounds of Formula I, II, III, IV, orV Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX

The CDK4/6 inhibitors for use in the described methods are highlyselective, potent CDK4/6 inhibitors, with minimal CDK2 inhibitoryactivity. In a range of embodiments, a compound for use in the methodsdescribed herein has a CDK4/CycD1 IC₅₀ inhibitory concentration valuethat is >1500 times, >1800 times, >2000 times, >2200 times, >2500times, >2700 times, >3000 times, >3200 times or greater lower than itsrespective IC₅₀ concentration value for CDK2/CycE inhibition. In anotherrange of embodiments, a compound for use in the methods described hereinhas an IC₅₀ concentration value for CDK4/CycD1 inhibition that is about<1.50 nM, <1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM,<0.70 nM, <0.65 nM, <0.60 nM, <0.55 nM, or less. In yet a range ofembodiments, a CDK4/6 inhibitor for use in the methods described hereinhas an IC₅₀ concentration value for CDK2/CycE inhibition that isabout >1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50μM, >2.75 μM, >3.0 μM, >3.25 μM, >3.5 μM or greater. In still otherembodiments, a compound for use in the methods described herein has anIC₅₀ concentration value for CDK2/CycA IC₅₀ that is >0.80 μM, >0.85μM, >0.90 μM, >0.95 μM, >0.1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0μM, >2.25 μM, >2.50 μM, >2.75 uM, >3.0 μM or greater.

Additional CDK4/6 inhibitors that may be useful in certain combinationsof the present invention include abemaciclib, palbociclib, andribociclib. Abemaciclib(N-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine)is described in U.S. Pat. No. 7,855,211 incorporated herein, and has thechemical structure:

Palbociclib(6-Acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one)is described in U.S. Pat. No. 7,208,489 incorporated herein, and has thechemical structure:

Ribociclib(7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide)is described in U.S. Pat. Application No. 2010/0105653 incorporatedherein, and has the chemical structure:

Topoisomerase I Inhibitors

DNA is normally a coiled double helix of two strands and is periodicallyuncoiled in the process of replication during cell division or in theprocess of reading the code to make protein. Two enzymes that play arole in this uncoiling and recoiling process are topoisomerase I andtopoisomerase II. They also play a significant role in fixing DNA damagethat occurs as a result of exposure to DNA damaging agents, for example,radiation exposure or chemotherapeutics.

Topoisomerase I attaches to DNA and breaks one strand of the doublehelix that can rotate around its own axis to revert supercoiling.Topoisomerase II binds to a DNA double strand and makes a gate allowinga second DNA double helix pass.

Topoisomerase I inhibitors target the enzyme topoisomerase I (Top1) bytrapping the catalytic intermediate of the Top1-DNA reaction, thecleavage complex. Topoisomerase I inhibitors, such as camptothecin, forma ternary complex, since they can trap the enzyme and DNA together

Many topoisomerase inhibitors are natural products extracted fromplants, such as camptothecin, isolated from Camptotheca accuminata, andseveral alkaloids, such as evodiamine, isolated from Evodia rutaecarpa.Topotecan and irinotecan are camptothecin derivatives that have beenused for ovarian and colorectal cancer treatments, respectively. Theseinhibitors target topo I and bind to DNA, forming a cleavable complex.The collision between this ternary complex and a replication forkgenerates DNA double-strand breaks, which may be related to the S-phasecytotoxicity, the G2/M cell cycle arrest and DNA damage that activatesrepair proteins.

In one aspect, an advantageous treatment of select Rb-negative cancersis disclosed using specific CDK4/6 inhibitor compounds in combinationwith a topoisomerase inhibitor. In one embodiment, the topoisomeraseinhibitor is a topoisomerase I inhibitor or a topoisomerase I and IIdual inhibitor. In one embodiment, the topoisomerase inhibitor is atopoisomerase II inhibitor.

In one embodiment, the topoisomerase inhibitor is selected from atopoisomerase I inhibitor. Known topoisomerase I inhibitors useful inthe present invention include(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dionemonohydrochloride (topotecan),(S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione(camptothecin),(1S,9S)-1-Amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo(de)pyrano(3′,4′:6,7)indolizino(1,2-b)quinoline-10,13-dione(exatecan),(7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin(lurtotecan), or(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′bipiperidine]-1′-carboxylate(irinotecan),(R)-5-ethyl-9,10-difluoro-5-hydroxy-4,5-dihydrooxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15(1H,13H)-dione(diflomotecan),(4S)-11-((E)-((1,1-Dimethylethoxy)imino)methyl)-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano(3′,4′:6,7)indolizino(1,2-b)quinoline-3,14(4H)-dione(gimatecan),(S)-8-ethyl-8-hydroxy-15-((4-methylpiperazin-1-yl)methyl)-11,14-dihydro-2H-[1,4]dioxino[2,3-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(3H,8H)-dione(lurtotecan),(4S)-4-Ethyl-4-hydroxy-11-[2-[(1-methylethyl)amino]ethyl]-1H-pyrano[3?,4?:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione(belotecan),6-((1,3-dihydroxypropan-2-yl)amino)-2,10-dihydroxy-12-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(edotecarin),8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxy-5H-dibenzo(c,h)(1,6)naphthyridin-6-one(topovale), benzo[6,7]indolizino[1,2-b]quinolin-11(13H)-one(rosettacin),(S)-4-ethyl-4-hydroxy-11-(2-(trimethylsilyl)ethyl)-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione(cositecan),tetrakis{(4S)-9-[([1,4′-bipiperidinyl]-1′-carbonyl)oxy]-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl}N,N′,N″,N′″-{methanetetrayltetrakis[methylenepoly(oxyethylene)oxy(1-oxoethylene)]}tetraglycinatetetrahydrochloride (etirinotecan pegol), 10-hydroxy-camptothecin(HOCPT), 9-nitrocamptothecin (rubitecan), SN38(7-ethyl-10-hydroxycamptothecin), and 10-hydroxy-9-nitrocamptothecin(CPT109),(R)-9-chloro-5-ethyl-5-hydroxy-10-methyl-12-((4-methylpiperidin-1-yl)methyl)-4,5-dihydrooxepino[3′,4′:6,7]indolizino[1,2-b]quinoline-3,15(1H,13H)-dione(elmotecan).

In a particular embodiment, the topoisomerase inhibitor is thetopoisomerase I inhibitor(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1Hpyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dionemonohydrochloride (topotecan hydrochloride).

In one embodiment of the invention, a CDK4/6 inhibitor is selected fromFormula I, II, III, IV, or V as described herein, abemaciclib,palbociclib, or ribociclib, and administered in a combination oralternation schedule with a topoisomerase inhibitor to a subject havingan Rb-negative cancer. In one embodiment of the invention, a CDK4/6inhibitor is selected from Formula I, II, III, IV, or V as describedherein, abemaciclib, palbociclib, or ribociclib, and administered in acombination or alternation schedule with a topoisomerase inhibitor to asubject having an Rb-negative gastric, glioma, breast, non-small celllung, small cell lung, esophageal, or liver cancer.

Furthermore, provided herein are methods and compositions for reducingthe development of secondary malignancies associated with the use oftopoisomerase inhibitors wherein a subject undergoing treatment for anRb-negative cancer with a topoisomerase inhibitor is provided a CDK4/6inhibitor described herein. In one embodiment, the topoisomeraseinhibitor is a topoisomerase I inhibitor. In one embodiment, thetopoisomerase inhibitor is a topoisomerase I and II dual inhibitor. Inone embodiment, the CDK4/6 inhibitor is selected from compounds Q, T, U,GG, X, and BB, or a combination thereof.

Isotopic Substitution

The present invention includes the use of CDK4/6 inhibitors incombination with topoisomerase I or II inhibitors for the treatment ofRb-negative proliferative disorders. In certain aspects, the compoundsused include compounds with desired isotopic substitutions of atoms, atamounts above the natural abundance of the isotope, i.e., enriched.Isotopes are atoms having the same atomic number but different massnumbers, i.e., the same number of protons but a different number ofneutrons. By way of general example and without limitation, isotopes ofhydrogen, for example, deuterium (²H) and tritium (³H) may be usedanywhere in the described structures. Alternatively or in addition,isotopes of carbon, e.g., ¹³C and ¹⁴C, may be used. A preferred isotopicsubstitution is deuterium for hydrogen at one or more locations on themolecule to improve the performance of the drug. The deuterium can bebound in a location of bond breakage during metabolism (an α-deuteriumkinetic isotope effect) or next to or near the site of bond breakage (aβ-deuterium kinetic isotope effect).

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein toa compound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The term “isotopic analog” refers to an analog that is a “deuteratedanalog”, a “¹³C-labeled analog,” or a “deuterated/¹³C-labeled analog.”The term “deuterated analog” means a compound described herein, wherebya H-isotope, i.e., hydrogen/protium (¹H), is substituted by a H-isotope,i.e., deuterium (²H). Deuterium substitution can be partial or complete.Partial deuterium substitution means that at least one hydrogen issubstituted by at least one deuterium. In certain embodiments, theisotope is 90, 95 or 99% or more enriched in an isotope at any locationof interest. In some embodiments it is deuterium that is 90, 95 or 99%enriched at a desired location.

Further specific isotopic analog compounds that fall within the presentinvention and that can be used in the disclosed methods of treatment andcompositions include the structures of Formula I, II, III, IV, or Vlisted in Table 1 above and/or the topoisomerase I inhibitors describedabove.

Rb-Negative Cancers and Proliferative Disorders

In particular, the active compounds described herein can be used incombination with one or more additional chemotherapeutic agents to treata subject suffering from a Rb-negative cancer or other Rb-negativeabnormal cellular proliferative disorder. Cancers and disorders of suchtype can be characterized by (e.g., that has cells that exhibit) theabsence of a functional Retinoblastoma protein. Such cancers anddisorders are classified as being Rb-negative. Rb-negative abnormalcellular proliferation disorders, and variations of this term as usedherein, refer to disorders or diseases caused by uncontrolled orabnormal cellular division which are characterized by the absence of afunctional Retinoblastoma protein, which can include cancers.

Such Rb-negative cancers can include, but are not limited to, small celllung cancer, retinoblastoma, HPV positive malignancies like cervicalcancer and certain head and neck cancers, MYC amplified tumors such asBurkitts' Lymphoma, and triple negative breast cancer; certain classesof sarcoma, for example Ewing's sarcoma, certain classes of non-smallcell lung carcinoma, certain classes of melanoma, certain classes ofpancreatic cancer, certain classes of leukemia, for example acutemyelogenous leukemia (AML) or acute lymphoblatic leukemia (ALL), certainclasses of lymphoma, certain classes of brain cancer, certain classes ofcolon cancer, certain classes of prostate cancer, certain classes ofovarian cancer, certain classes of uterine cancer, certain classes ofthyroid and other endocrine tissue cancers, certain classes of salivarycancers, certain classes of thymic carcinomas, certain classes of kidneycancers, certain classes of bladder cancers, and certain classes oftesticular cancers. In a specific embodiment, the Rb-negative leukemiais acute myeloid leukemia. In additional nonlimiting embodiments, thehost may be suffering from an acute or chronic Rb-negative leukemia of alymphocytic or myelogenous origin, such as, but not limited to: Acutelymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML); Chroniclymphocytic leukemia (CLL); Chronic myelogenous leukemia (CML); juvenilemyelomonocytic leukemia (JMML); hairy cell leukemia (HCL); acutepromyelocytic leukemia (a subtype of AML); large granular lymphocyticleukemia; or Adult T-cell chronic leukemia. In one embodiment, thepatient suffers from an acute myelogenous leukemia, for example anundifferentiated AML (M0); myeloblastic leukemia (M1; with/withoutminimal cell maturation); myeloblastic leukemia (M2; with cellmaturation); promyelocytic leukemia (M3 or M3 variant [M3V]);myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]);monocytic leukemia (M5); erythroleukemia (M6); or megakaryoblasticleukemia (M7).

The presence or absence of a normal functioning of the retinoblastoma(Rb) tumor suppressor protein can be determined through any of thestandard assays known to one of ordinary skill in the art, including butnot limited to Western Blot, ELISA (enzyme linked immunoadsorbentassay), IHC (immunohistochemistry), and FACS (fluorescent activated cellsorting). The selection of the assay will depend upon the tissue, cellline or surrogate tissue sample that is utilized e.g., for exampleWestern Blot and ELISA may be used with any or all types of tissues,cell lines or surrogate tissues, whereas the IHC method would be moreappropriate wherein the tissue utilized in the methods of the presentinvention was a tumor biopsy. FACs analysis would be most applicable tosamples that were single cell suspensions such as cell lines andisolated peripheral blood mononuclear cells. See for example, US20070212736 “Functional Immunohistochemical Cell Cycle Analysis as aPrognostic Indicator for Cancer”. Alternatively, molecular genetictesting may be used for determination of retinoblastoma gene status.Molecular genetic testing for retinoblastoma includes the following asdescribed in Lohmann and Gallie “Retinoblastoma. Gene Reviews” (2010)http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=retinoblastomaor Parsam et al. “A comprehensive, sensitive and economical approach forthe detection of mutations in the RB 1 gene in retinoblastoma” Journalof Genetics, 88(4), 517-527 (2009).

In a specific embodiment, the Rb-negative cancer is small cell lungcarcinoma.

In a specific embodiment, the Rb-negative cancer is cervical cancer.

In a specific embodiment, the Rb-negative cancer is neuroblastoma.

In a specific embodiment, the Rb-negative cancer is a sarcoma, forexample, Ewing sarcoma.

In a specific embodiment, the Rb-negative cancer is an ovarian cancer.

In a specific embodiment, the Rb-negative cancer is a gastric cancer.

In a specific embodiment, the Rb-negative cancer is glioblastoma.

In a specific embodiment, the Rb-negative cancer is breast cancer.

In a specific embodiment, the Rb-negative cancer is triple-negativebreast cancer.

In a specific embodiment, the Rb-negative cancer is a non-small celllung cancer.

In a specific embodiment, the Rb-negative cancer is an esophagealcancer.

In a specific embodiment, the Rb-negative cancer is Burkitts' Lymphoma.

In a specific embodiment, the Rb-negative cancer is a melanoma.

In a specific embodiment, the Rb-negative cancer is pancreatic cancer.

In a specific embodiment, the Rb-negative cancer is leukemia, forexample acute myelogenous leukemia (AML) or acute lymphoblastic leukemia(ALL).

In a specific embodiment, the Rb-negative cancer is brain cancer.

In a specific embodiment, the Rb-negative cancer is colon cancer.

In a specific embodiment, the Rb-negative cancer is prostate cancer.

In a specific embodiment, the Rb-negative cancer is uterine cancer.

In a specific embodiment, the Rb-negative cancer is thyroid and otherendocrine tissue cancers.

In a specific embodiment, the Rb-negative cancer is salivary cancers.

In a specific embodiment, the Rb-negative cancer is a thymic carcinoma.

In a specific embodiment, the Rb-negative cancer is kidney cancer.

In a specific embodiment, the Rb-negative cancer is bladder cancer.

In a specific embodiment, the Rb-negative cancer is testicular cancer.

The methods of treatment described herein can be used in furthercombination with one or more additional chemotherapeutic agents to treata subject suffering from an Rb-negative cancer.

Combination Therapy

In one aspect of the invention, the combination of a CDK4/6 inhibitordescribed herein and a topoisomerase I inhibitor to treat an Rb-negativecellular proliferation disorder can be further beneficially administeredin combination with another therapeutic regimen for beneficial,additive, or synergistic effect.

The additional therapy can be an immunotherapy. In another embodiment,the combination is used with another pharmaceutical or a biologic agent(for example an antibody) to increase the efficacy of treatment with acombined or a synergistic approach. In an embodiment, the CDK4/6inhibitor and topoisomerase I inhibitor can be used with T-cellvaccination, which typically involves immunization with inactivatedauto-reactive T cells to eliminate an Rb-negative cancer cell populationas described herein. In another embodiment, the combination is used infurther combination with a bispecific T-cell Engager (BiTE), which is anantibody designed to simultaneously bind to specific antigens onendogenous T cells and Rb-negative cancer cells as described herein,linking the two types of cells.

In one embodiment, the additional therapy is a monoclonal antibody(MAb). Some MAbs stimulate an immune response that destroys cancercells. Similar to the antibodies produced naturally by B cells, theseMAbs “coat” the cancer cell surface, triggering its destruction by theimmune system. For example, bevacizumab targets vascular endothelialgrowth factor (VEGF), a protein secreted by tumor cells and other cellsin the tumor's microenvironment that promotes the development of tumorblood vessels. When bound to bevacizumab, VEGF cannot interact with itscellular receptor, preventing the signaling that leads to the growth ofnew blood vessels. Similarly, cetuximab and panitumumab target theepidermal growth factor receptor (EGFR), and trastuzumab targets thehuman epidermal growth factor receptor 2 (HER-2). MAbs that bind to cellsurface growth factor receptors prevent the targeted receptors fromsending their normal growth-promoting signals. They may also triggerapoptosis and activate the immune system to destroy tumor cells.

Another group of cancer therapeutic MAbs are the immunoconjugates. TheseMAbs, which are sometimes called immunotoxins or antibody-drugconjugates, consist of an antibody attached to a cell-killing substance,such as a plant or bacterial toxin, a chemotherapy drug, or aradioactive molecule. The antibody latches onto its specific antigen onthe surface of a cancer cell, and the cell-killing substance is taken upby the cell. FDA-approved conjugated MAbs that work this way includeado-trastuzumab emtansine, which targets the HER-2 molecule to deliverthe drug DM1, which inhibits cell proliferation, to HER-2 expressingmetastatic breast cancer cells.

Immunotherapies with T cells engineered to recognize cancer cells viabispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) areapproaches with potential to ablate both dividing and non/slow-dividingsubpopulations of cancer cells.

Bispecific antibodies, by simultaneously recognizing target antigen andan activating receptor on the surface of an immune effector cell, offeran opportunity to redirect immune effector cells to kill cancer cells.The other approach is the generation of chimeric antigen receptors byfusing extracellular antibodies to intracellular signaling domains.Chimeric antigen receptor-engineered T cells are able to specificallykill tumor cells in a MHC-independent way.

In some embodiments, the combination can be administered to the subjectin further combination with an additional chemotherapeutic agent. In oneembodiment, the use of the compounds described herein is combined in atherapeutic regime with other agents.

Further additional chemotherapeutic agents may include, but are notlimited to, tamoxifen, midazolam, letrozole, bortezomib, anastrozole,goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dual mTOR-PI3Kinhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors, HSPinhibitors (for example, HSP70 and HSP 90 inhibitors, or a combinationthereof), BCL-2 inhibitors, apopototic inducing compounds, AKTinhibitors, including but not limited to, MK-2206, GSK690693,Perifosine, (KRX-0401), GDC-0068, Triciribine, AZD5363, Honokiol,PF-04691502, and Miltefosine, PD-1 inhibitors including but not limitedto, Nivolumab, CT-011, MK-3475, BMS936558, and AMP-514 or FLT-3inhibitors, including but not limited to, P406, Dovitinib, Quizartinib(AC220), Amuvatinib (MP-470), Tandutinib (MLN518), ENMD-2076, andKW-2449, or combinations thereof. Examples of mTOR inhibitors includebut are not limited to rapamycin and its analogs, everolimus (Afinitor),temsirolimus, ridaforolimus, sirolimus, and deforolimus. Examples of P13kinase inhibitors include but are not limited to Wortmannin,demethoxyviridin, perifosine, idelalisib, PX-866, IPI-145, BAY 80-6946,BEZ235, RP6503, TGR 1202 (RP5264), MLN1117 (INK1117), Pictilisib,Taselisib (GDC-0032), Buparlisib, SAR245408 (XL147), SAR245409 (XL765),Palomid 529, ZSTK474, PWT33597, RP6530, CUDC-907, and AEZS-136. Examplesof MEK inhibitors include but are not limited to Trametinib,Selumetinib, MEK162, GDC-0973 (XL518), and PD0325901. Examples of RASinhibitors include but are not limited to Reolysin and siG12D LODER.Examples of ALK inhibitors include but are not limited to Crizotinib,Ceritinib (Zykadia), AP26113, and LDK378. HSP inhibitors include but arenot limited to Geldanamycin or 17-N-Allylamino-17-demethoxygeldanamycin(17AAG), and Radicicol. In a particular embodiment, a compound describedherein is administered in combination with letrozole and/or tamoxifen.Other chemotherapeutic agents that can be used in combination with thecompounds described herein include, but are not limited to,chemotherapeutic agents that do not require cell cycle activity fortheir anti-neoplastic effect.

In one embodiment, the CDK4/6 inhibitor and topoisomerase I inhibitorcan be further combined with a chemotherapeutic selected from, but arenot limited to, Imatinib mesylate (Gleevac®), Dasatinib (Sprycel®),Nilotinib (Tasigna®), Bosutinib (Bosulif®), Trastuzumab (Herceptin®),trastuzumab-DM1, Pertuzumab (Perjeta™), Lapatinib (Tykerb®), Gefitinib(Iressa®), Erlotinib (Tarceva®), Cetuximab (Erbitux®), Panitumumab(Vectibix®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat(Zolinza®), Romidepsin (Istodax®), Bexarotene (Tagretin®), Alitretinoin(Panretin®), Tretinoin (Vesanoid®), Carfilizomib (Kyprolis™),Pralatrexate (Folotyn®), Bevacizumab (Avastin®), Ziv-aflibercept(Zaltrap®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Pazopanib(Votrient®), Regorafenib (Stivarga®), and Cabozantinib (Cometriq™).

In certain aspects, the additional therapeutic agent is ananti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic,additional therapeutic agents, or immunosuppressive agents.

Suitable chemotherapeutic agents include, but are not limited to,radioactive molecules, toxins, also referred to as cytotoxins orcytotoxic agents, which includes any agent that is detrimental to theviability of cells, agents, and liposomes or other vesicles containingchemotherapeutic compounds. General anticancer pharmaceutical agentsinclude: Vincristine (Oncovin®) or liposomal vincristine (Marqibo®),Daunorubicin (daunomycin or Cerubidine®) or doxorubicin (Adriamycin®),Cytarabine (cytosine arabinoside, ara-C, or Cytosar®), L-asparaginase(Elspar®) or PEG-L-asparaginase (pegaspargase or Oncaspar®), Etoposide(VP-16), Teniposide (Vumon®), 6-mercaptopurine (6-MP or Purinethol®),Methotrexate, Cyclophosphamide (Cytoxan®), Prednisone, Dexamethasone(Decadron), imatinib (Gleevec®), dasatinib (Sprycel®), nilotinib(Tasigna®), bosutinib (Bosulif®), and ponatinib (Iclusig™). Examples ofadditional suitable chemotherapeutic agents include but are not limitedto 1-dehydrotestosterone, 5-fluorouracil, dacarbazine, 6-mercaptopurine,6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylatingagents, allopurinol sodium, altretamine, amifostine, anastrozole,anthramycin (AMC)), anti-mitotic agents, cis-dichlorodiamine platinum(II) (DDP) cisplatin), diamino dichloro platinum, anthracyclines,antibiotics, antimetabolites, asparaginase, BCG live (intravesical),betamethasone sodium phosphate and betamethasone acetate, bicalutamide,bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin,capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU),Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens,Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasinB, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin (formerlyactinomycin), daunirubicin HCL, daunorucbicin citrate, denileukindiftitox, Dexrazoxane, Dibromomannitol, dihydroxy anthracin dione,Docetaxel, dolasetron mesylate, doxorubicin HCL, dronabinol, E. coliL-asparaginase, emetine, epoetin-α, Erwinia L-asparaginase, esterifiedestrogens, estradiol, estramustine phosphate sodium, ethidium bromide,ethinyl estradiol, etidronate, etoposide citrororum factor, etoposidephosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate,fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids,goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea,idarubicin HCL, ifosfamide, interferon α-2b, letrozole, leucovorincalcium, leuprolide acetate, levamisole HCL, lidocaine, lomustine,maytansinoid, mechlorethamine HCL, medroxyprogesterone acetate,megestrol acetate, melphalan HCL, mercaptipurine, mesna, methotrexate,methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone,nilutamide, octreotide acetate, ondansetron HCL, paclitaxel, pamidronatedisodium, pentostatin, pilocarpine HCL, plimycin, polifeprosan 20 withcarmustine implant, porfimer sodium, procaine, procarbazine HCL,propranolol, rituximab, sargramostim, streptozotocin, tamoxifen, taxol,teniposide, tenoposide, testolactone, tetracaine, thioepa chlorambucil,thioguanine, thiotepa, topotecan HCL, toremifene citrate, trastuzumab,tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, andvinorelbine tartrate.

Additional therapeutic agents that can be administered in combinationwith a compound disclosed herein can include bevacizumab, sutinib,sorafenib, 2-methoxyestradiol or 2ME2, finasunate, vatalanib,vandetanib, aflibercept, volociximab, etaracizumab (MEDI-522),cilengitide, erlotinib, cetuximab, panitumumab, gefitinib, trastuzumab,dovitinib, figitumumab, atacicept, rituximab, alemtuzumab, aldesleukine,atlizumab, tocilizumab, temsirolimus, everolimus, lucatumumab,dacetuzumab, HLL1, huN901-DM1, atiprimod, natalizumab, bortezomib,carfilzomib, marizomib, tanespimycin, saquinavir mesylate, ritonavir,nelfinavir mesylate, indinavir sulfate, belinostat, panobinostat,mapatumumab, lexatumumab, dulanermin, ABT-737, oblimersen, plitidepsin,talmapimod, P276-00, enzastaurin, tipifarnib, perifosine, imatinib,dasatinib, lenalidomide, thalidomide, simvastatin, celecoxib,bazedoxifene, AZD4547, rilotumumab, oxaliplatin (Eloxatin), palbociclib(PD0332991), ribociclib (LEEO11), abemaciclib (LY2835219), HDM201,fulvestrant (Faslodex), exemestane (Aromasin), PIM447, ruxolitinib(INC424), BGJ398, necitumumab, pemetrexed (Alimta), and ramucirumab(IMC-1121B).

Further treatment regimen includes the administration of at least oneadditional kinase inhibitor. In one embodiment, the at least oneadditional kinase inhibitor is selected from a phosphoinositide 3-kinase(PI3K) inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, or aspleen tyrosine kinase (Syk) inhibitor, or a combination thereof.

PI3K inhibitors that may be used in the present invention are wellknown. Examples of PI3 kinase inhibitors include but are not limited toWortmannin, demethoxyviridin, perifosine, idelalisib, Pictilisib,Palomid 529, ZSTK474, PWT33597, CUDC-907, and AEZS-136, duvelisib,GS-9820, BKM120, GDC-0032 (Taselisib)(2-[4-[2-(2-Isopropyl-5-methyl-1,2,4-triazol-3-yl)-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin-9-yl]pyrazol-1-yl]-2-methylpropanamide),MLN-1117 ((2R)-1-Phenoxy-2-butanyl hydrogen (S)-methylphosphonate; orMethyl(oxo) {[(2R)-1-phenoxy-2-butanyl]oxy}phosphonium)), BYL-719((2S)—N1-[4-Methyl-5-[2-(2,2,2-trifluoro-1,1-dimethylethyl)-4-pyridinyl]-2-thiazolyl]-1,2-pyrrolidinedicarboxamide),GSK2126458(2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide)(omipalisib), TGX-221((±)-7-Methyl-2-(morpholin-4-yl)-9-(1-phenylaminoethyl)-pyrido[1,2-a]-pyrimidin-4-one),GSK2636771(2-Methyl-1-(2-methyl-3-(trifluoromethyl)benzyl)-6-morpholino-1H-benzo[d]imidazole-4-carboxylicacid dihydrochloride), KIN-193((R)-2-((1-(7-methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)ethyl)amino)benzoicacid), TGR-1202/RP5264, GS-9820((S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-mohydroxypropan-1-one),GS-1101(5-fluoro-3-phenyl-2-([S)]-1-[9H-purin-6-ylamino]-propyl)-3H-quinazolin-4-one),AMG-319, GSK-2269557, SAR245409(N-(4-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4methylbenzamide), BAY80-6946(2-amino-N-(7-methoxy-8-(3-morpholinopropoxy)-2,3-dihydroimidazo[1,2-c]quinaz),AS 252424(5-[1-[5-(4-Fluoro-2-hydroxy-phenyl)-furan-2-yl]-meth-(Z)-ylidene]-thiazolidine-2,4-dione),CZ 24832(5-(2-amino-8-fluoro-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide),Buparlisib(5-[2,6-Di(4-morpholinyl)-4-pyrimidinyl]-4-(trifluoromethyl)-2-pyridinamine),GDC-0941(2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)-1-piperazinyl]methyl]-4-(4-morpholinyl)thieno[3,2-d]pyrimidine),GDC-0980((S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one (also known as RG7422)),SF1126((8S,14S,17S)-14-(carboxymethyl)-8-(3-guanidinopropyl)-17-(hydroxymethyl)-3,6,9,12,15-pentaoxo-1-(4-(4-oxo-8-phenyl-4H-chromen-2-yl)morpholino-4-ium)-2-oxa-7,10,13,16-tetraazaoctadecan-18-oate),PF-05212384(N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N′-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea)(gedatolisib), LY3023414, BEZ235(2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile)(dactolisib), XL-765(N-(3-(N-(3-(3,5-dimethoxyphenylamino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide),and GSK1059615(5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione),PX886([(3aR,6E,9S,9aR,10R,11aS)-6-[[bis(prop-2-enyl)amino]methylidene]-5-hydroxy-9-(methoxymethyl)-9a,11a-dimethyl-1,4,7-trioxo-2,3,3a,9,10,11-hexahydroindeno[4,5h]isochromen-10-yl]acetate (also known as sonolisib)), LY294002, AZD8186, PF-4989216,pilaralisib, GNE-317, PI-3065, PI-103, NU7441 (KU-57788), HS 173,VS-5584 (SB2343), CZC24832, TG100-115, A66, YM201636, CAY10505, PIK-75,PIK-93, AS-605240, BGT226 (NVP-BGT226), AZD6482, voxtalisib, alpelisib,IC-87114, TGI100713, CH5132799, PKI-402, copanlisib (BAY 80-6946), XL147, PIK-90, PIK-293, PIK-294, 3-MA (3-methyladenine), AS-252424,AS-604850, apitolisib (GDC-0980; RG7422), and the structure described inWO2014/071109 having the formula:

In one embodiment, the CDK4/6 inhibitor is combined in a single dosageform with the PIk3 inhibitor.

BTK inhibitors for use in the present invention are well known. Examplesof BTK inhibitors include ibrutinib (also known as PCI-32765)(Imbruvica™)(1-[(3R)-3-[4-amino-3-(4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one),dianilinopyrimidine-based inhibitors such as AVL-101 and AVL-291/292(N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide)(Avila Therapeutics) (see US Patent Publication No 2011/0117073,incorporated herein in its entirety), Dasatinib([N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide],LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-ibromophenyl)propenamide), GDC-0834([R—N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide],CGI-5604-(tert-butyl)-N-(3-(8-(phenylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)benzamide,CGI-1746(4-(tert-butyl)-N-(2-methyl-3-(4-methyl-6-((4-(morpholine-4-carbonyl)phenyl)amino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide),CNX-774(4-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenoxy)-N-methylpicolinamide),CTA056(7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one),GDC-0834((R)—N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl-1-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide),GDC-0837((R)—N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl-1-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide),HM-71224, ACP-196, ONO-4059 (Ono Pharmaceuticals), PRT062607(4-((3-(2H-1,2,3-triazol-2-yl)phenyl)amino)-2-(((1R,2S)-2-aminocyclohexyl)amino)pyrimidine-5-carboxamidehydrochloride), QL-47(1-(1-acryloylindolin-6-yl)-9-(1-methyl-1H-pyrazol-4-yl)benzo[h][1,6]naphthyridin-2(1H)-one),and RN486(6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one),and other molecules capable of inhibiting BTK activity, for examplethose BTK inhibitors disclosed in Akinleye et ah, Journal of Hematology& Oncology, 2013, 6:59, the entirety of which is incorporated herein byreference. In one embodiment, the CDK4/6 inhibitor is combined in asingle dosage form with the BTK inhibitor.

Syk inhibitors for use in the present invention are well known, andinclude, for example, Cerdulatinib(4-(cyclopropylamino)-2-((4-(4-(ethylsulfonyl)piperazin-1-yl)phenyl)amino)pyrimidine-5-carboxamide),entospletinib(6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine),fostamatinib([6-({5-Fluoro-2-[(3,4,5-trimethoxyphenyl)amino]-4-pyrimidinyl}amino)-2,2-dimethyl-3-oxo-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazin-4-yl]methyldihydrogen phosphate), fostamatinib disodium salt (sodium(6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-3-oxo-2H-pyrido[3,2-b][1,4]oxazin-4(3H)-yl)methylphosphate), BAY 61-3606(2-(7-(3,4-Dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino)-nicotinamideHCl), RO9021(6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylicacid amide), imatinib (Gleevac;4-[(4-methylpiperazin-1-yl)methyl]-N-(4-methyl-3-{[4-(pyridin-3-yl)pyrimidin-2-yl]amino}phenyl)benzamide),staurosporine, GSK143(2-(((3R,4R)-3-aminotetrahydro-2H-pyran-4-yl)amino)-4-(p-tolylamino)pyrimidine-5-carboxamide),PP2(1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine),PRT-060318(2-(((1R,2S)-2-aminocyclohexyl)amino)-4-(m-tolylamino)pyrimidine-5-carboxamide),PRT-062607(4-((3-(2H-1,2,3-triazol-2-yl)phenyl)amino)-2-(((1R,2S)-2-aminocyclohexyl)amino)pyrimidine-5-carboxamidehydrochloride), R112(3,3′-((5-fluoropyrimidine-2,4-diyl)bis(azanediyl))diphenol), R348(3-Ethyl-4-methylpyridine), R406(6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one), piceatannol (3-Hydroxyresveratol), YM193306 (see Singh et al.Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J.Med. Chem. 2012, 55, 3614-3643), 7-azaindole, piceatannol, ER-27319 (seeSingh et al. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), Compound D (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), PRT060318 (see Singh etal. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), luteolin (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), apigenin (see Singh etal. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), quercetin (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), fisetin (see Singh etal. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), myricetin (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), morin (see Singh et al.Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J.Med. Chem. 2012, 55, 3614-3643 incorporated in its entirety herein). Inone embodiment, the CDK4/6 inhibitor is combined in a single dosage formwith the Syk inhibitor.

MEK inhibitors for use in the present invention are well known, andinclude, for example, trametinib/GSK1120212(N-(3-{3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H-yl}phenyl)acetamide),selumetinib(6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide),pimasertib/AS703026/MSC 1935369((S)—N-(2,3-dihydroxypropyl)-3-((2-fluoro-4-iodophenyl)amino)isonicotinamide),XL-518/GDC-0973(1-({3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl}carbonyl)-3-[(2S)-piperidin-2-yl]azetidin-3-ol),refametinib/BAY869766/RDEAl 19(N-(3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-6-methoxyphenyl)-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide),PD-0325901(N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide),TAK733((R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione),MEK162/ARRY438162(5-[(4-Bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide),R05126766 (3-[[3-Fluoro-2-(methylsulfamoylamino)-4-pyridyl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one),WX-554, R04987655/CH4987655(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-5-((3-oxo-1,2-oxazinan-2yl)methyl)benzamide),or AZD8330 (2-((2-fluoro-4-iodophenyl)amino)-N-(2hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide),U0126-EtOH, PD184352 (CI-1040), GDC-0623, BI-847325, cobimetinib,PD98059, BIX 02189, BIX 02188, binimetinib, SL-327, TAK-733, PD318088,and additional MEK inhibitors as described below. In one embodiment, theCDK4/6 inhibitor is combined in a single dosage form with the MEKinhibitor.

Raf inhibitors for use in the present invention are well known, andinclude, for example, Vemurafinib(N-[3-[[5-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl]-2,4-difluorophenyl]-1-propanesulfonamide),sorafenib tosylate(4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide;4-methylbenzenesulfonate), AZ628(3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin-6-ylamino)phenyl)benzamide),NVP-BHG712(4-methyl-3-(1-methyl-6-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-N-(3-(trifluoromethyl)phenyl)benzamide),RAF-265(1-methyl-5-[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]pyridin-4-yl]oxy-N-[4-(trifluoromethyl)phenyl]benzimidazol-2-amine),2-Bromoaldisine(2-Bromo-6,7-dihydro-1H,5H-pyrrolo[2,3-c]azepine-4,8-dione), Raf KinaseInhibitor IV(2-chloro-5-(2-phenyl-5-(pyridin-4-yl)-1H-imidazol-4-yl)phenol),Sorafenib N-Oxide (4-[4-[[[[4-Chloro-3(trifluoroMethyl)phenyl]aMino]carbonyl]aMino]phenoxy]-N-Methyl-2pyridinecarboxaMide1-Oxide), PLX-4720, dabrafenib (GSK2118436), GDC-0879, RAF265, AZ 628,SB590885, ZM336372, GW5074, TAK-632, CEP-32496, LY3009120, and GX818(Encorafenib). In one embodiment, the CDK4/6 inhibitor is combined in asingle dosage form with the Raf inhibitor.

In one embodiment, the at least one additional chemotherapeutic agentcombined or alternated with the CDK4/6 inhibitor is a programmed deathprotein 1 (PD-1) inhibitor or programmed death protein ligand 1 or 2inhibitor. PD-1 inhibitors are known in the art, and include, forexample, nivolumab (BMS), pembrolizumab (Merck), pidilizumab(CureTech/Teva), AMP-244 (Amplimmune/GSK), BMS-936559 (BMS), andMEDI4736 (Roche/Genentech), and MPDL3280A (Genentech). In oneembodiment, the CDK4/6 inhibitor is combined in a single dosage formwith the PD-1 inhibitor.

In one embodiment, the at least one additional chemotherapeutic agentcombined or alternated with the CDK4/6 inhibitor is a B-cell lymphoma 2(Bcl-2) protein inhibitor. BCL-2 inhibitors are known in the art, andinclude, for example, ABT-199(4-[4-[[2-(4-Chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl]piperazin-1-yl]-N-[[3-nitro-4-[[(tetrahydro-2H-pyran-4-yl)methyl]amino]phenyl]sulfonyl]-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide),ABT-737(4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) (navitoclax), ABT-263((R)-4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio)butan-2-yl)amino)-3((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide),GX15-070 (obatoclax mesylate,(2Z)-2-[(5Z)-5-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-4-methoxypyrrol-2-ylidene]indole;methanesulfonic acid))), 2-methoxy-antimycin A3, YC137(4-(4,9-dioxo-4,9-dihydronaphtho[2,3-d]thiazol-2-ylamino)-phenyl ester),pogosin, ethyl2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate,Nilotinib-d3, TW-37(N-[4-[[2-(1,1-Dimethylethyl)phenyl]sulfonyl]phenyl]-2,3,4-trihydroxy-5-[[2-(1-methylethyl)phenyl]methyl]benzamide),Apogossypolone (ApoG2), HA14-1, AT101, sabutoclax, gambogic acid, orG3139 (Oblimersen). In one embodiment, the CDK4/6 inhibitor is combinedin a single dosage form with the at least one BCL-2 inhibitor.

In one aspect of the present invention, the combination therapydescribed herein can be additionally combined with at least oneimmunosuppressive agent. The immunosuppressive agent is preferablyselected from the group consisting of a calcineurin inhibitor, e.g. acyclosporin or an ascomycin, e.g. Cyclosporin A (NEORAL®), FK506(tacrolimus), pimecrolimus, a mTOR inhibitor, e.g. rapamycin or aderivative thereof, e.g. Sirolimus (RAPAMUNE®), Everolimus (Certican®),temsirolimus, zotarolimus, biolimus-7, biolimus-9, a rapalog,e.g.ridaforolimus, azathioprine, campath 1H, a S1P receptor modulator,e.g. fingolimod or an analogue thereof, an anti IL-8 antibody,mycophenolic acid or a salt thereof, e.g. sodium salt, or a prodrugthereof, e.g. Mycophenolate Mofetil (CELLCEPT®), OKT3 (ORTHOCLONEOKT3®), Prednisone, ATGAM®, THYMOGLOBULIN®, Brequinar Sodium, OKT4,T10B9.A-3A, 33B3.1, 15-deoxyspergualin, tresperimus, Leflunomide ARAVA®,CTLAI-Ig, anti-CD25, anti-IL2R, Basiliximab (SIMULECT®), Daclizumab(ZENAPAX®), mizorbine, methotrexate, dexamethasone, ISAtx-247, SDZ ASM981 (pimecrolimus, Elidel®), CTLA4lg (Abatacept), belatacept, LFA3lg,etanercept (sold as Enbrel® by Immunex), adalimumab (Humira®),infliximab (Remicade®), an anti-LFA-1 antibody, natalizumab (Antegren®),Enlimomab, gavilimomab, antithymocyte immunoglobulin, siplizumab,Alefacept efalizumab, pentasa, mesalazine, asacol, codeine phosphate,benorylate, fenbufen, naprosyn, diclofenac, etodolac and indomethacin,aspirin and ibuprofen.

In certain embodiments, the combination treatment described herein isadministered to the subject prior to treatment with anotherchemotherapeutic agent, during treatment with another chemotherapeuticagent, after administration of another chemotherapeutic agent, or acombination thereof.

Drug Conjugates

In one embodiment, the activity of the CDK4/6 inhibitor and/ortopoisomerase I inhibitor can be augmented through conjugation to anagent that targets the diseased or abnormally proliferating cell orotherwise enhances activity, delivery, pharmacokinetics or otherbeneficial property.

For example, the CDK4/6 inhibitor and/or topoisomerase I inhibitor canbe administered as an antibody-drug conjugates (ADC). In certainembodiments, a CDK4/6 inhibitor and/or topoisomerase I inhibitor can beadministered in conjugation or combination with an antibody or antibodyfragment. Fragments of an antibody can be produced through chemical orgenetic mechanisms. The antibody fragment can be an antigen bindingfragment. For example, the antigen binding fragment can be selected froman Fab, Fab′, (Fab′)2, or Fv. The antibody fragment can be a Fab.Monovalent F(ab) fragments have one antigen binding site. The antibodycan be a divalent (Fab′)2 fragment, which has two antigen bindingregions that are linked by disulfide bonds. In one embodiment, theantigen fragment is a (Fab′). Reduction of F(ab′)2 fragments producestwo monovalent Fab′ fragments, which have a free sulfhydryl group thatis useful for conjugation to other molecules.

A CDK4/6 inhibitor and/or topoisomerase I inhibitor described herein canbe administered in conjugation or combination with a Fv fragment. Fvfragments are the smallest fragment made from enzymatic cleavage of IgGand IgM class antibodies. Fv fragments have the antigen-binding sitemade of the VH and VC regions, but they lack the CH1 and CL regions. TheVH and VL chains are held together in Fv fragments by non-covalentinteractions.

In one embodiment, a CDK4/6 inhibitor and/or topoisomerase I inhibitoras described herein can be administered in combination with an antibodyfragment selected from the group consisting of an ScFv, domain antibody,diabody, triabody, tetrabody, Bis-scFv, minibody, Fab2, or Fab3 antibodyfragment. In one embodiment, the antibody fragment is a ScFv. Geneticengineering methods allow the production of single chain variablefragments (ScFv), which are Fv type fragments that include the VH and VLdomains linked with a flexible peptide When the linker is at least 12residues long, the ScFv fragments are primarily monomeric. Manipulationof the orientation of the V-domains and the linker length createsdifferent forms of Fv molecules. Linkers that are 3-11 residues longyield scFv molecules that are unable to fold into a functional Fvdomain. These molecules can associate with a second scFv molecule, tocreate a bivalent diabody. In one embodiment, the antibody fragmentadministered in combination with a CDK4/6 inhibitor and/or topoisomeraseI inhibitor described herein is a bivalent diabody. If the linker lengthis less than three residues, scFv molecules associate into triabodies ortetrabodies. In one embodiment, the antibody fragment is a triabody. Inone embodiment, the antibody fragment is a tetrabody. Multivalent scFvspossess greater functional binding affinity to their target antigensthan their monovalent counterparts by having binding to two more targetantigens, which reduces the off-rate of the antibody fragment. In oneembodiment, the antibody fragment is a minibody. Minibodies are scFv-CH3fusion proteins that assemble into bivalent dimers. In one embodiment,the antibody fragment is a Bis-scFv fragment. Bis-scFv fragments arebispecific. Miniaturized ScFv fragments can be generated that have twodifferent variable domains, allowing these Bis-scFv molecules toconcurrently bind to two different epitopes.

In one embodiment, a CDK4/6 inhibitor and/or topoisomerase I inhibitordescribed herein is administered in conjugation or combination with abispecific dimer (Fab2) or trispecific dimer (Fab3). Genetic methods arealso used to create bispecific Fab dimers (Fab2) and trispecific Fabtrimers (Fab3). These antibody fragments are able to bind 2 (Fab2) or 3(Fab3) different antigens at once.

In one embodiment, a CDK4/6 inhibitor and/or topoisomerase I inhibitordescribed herein is administered in conjugation or combination with anrIgG antibody fragment. rIgG antibody fragments refers to reduced IgG(75,000 daltons) or half-IgG. It is the product of selectively reducingjust the hinge-region disulfide bonds. Although several disulfide bondsoccur in IgG, those in the hinge-region are most accessible and easiestto reduce, especially with mild reducing agents like2-mercaptoethylamine (2-MEA). Half-IgG are frequently prepared for thepurpose of targeting the exposing hinge-region sulfhydryl groups thatcan be targeted for conjugation, either antibody immobilization orenzyme labeling.

In other embodiments, a CDK4/6 inhibitor and/or topoisomerase Iinhibitor described herein can be linked to a radioisotope to increaseefficacy, using methods well known in the art. Any radioisotope that isuseful against Rb-negative cancer cells can be incorporated into theconjugate, for example, but not limited to, ¹³¹I, ¹²³I, ¹⁹²Ir, ³²P,⁹⁰Sr, ¹⁹⁸Au, ²²⁶Ra, ⁹⁰Y, ²⁴¹Am, ²⁵²Cf, ⁶⁰Co, or ¹³⁷Cs.

Of note, the linker chemistry can be important to efficacy andtolerability of the drug conjugates. The thio-ether linked T-DM1increases the serum stability relative to a disulfide linker version andappears to undergo endosomal degradation, resulting in intra-cellularrelease of the cytotoxic agent, thereby improving efficacy andtolerability, See, Barginear, M. F. and Budman, D. R., Trastuzumab-DM1:A review of the novel immune-conjugate for HER2-overexpressing breastcancer, The Open Breast Cancer Journal, 1:25-30, 2009.

Examples of early and recent antibody-drug conjugates, discussing drugs,linker chemistries and classes of targets for product development thatmay be used in the present invention can be found in the reviews byCasi, G. and Neri, D., Antibody-drug conjugates: basic concepts,examples and future perspectives, J. Control Release 161(2):422-428,2012, Chari, R. V., Targeted cancer therapy: conferring specificity tocytotoxic drugs, Acc. Chem. Rev., 41(1):98-107, 2008, Sapra, P. andShor, B., Monoclonal antibody-based therapies in cancer: advances andchallenges, Pharmacol. Ther., 138(3):452-69, 2013, Schliemann, C. andNeri, D., Antibody-based targeting of the tumor vasculature, Biochim.Biophys. Acta., 1776(2):175-92, 2007, Sun, Y., Yu, F., and Sun, B. W.,Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue XueBao, 44(9):943-52, 2009, Teicher, B. A., and Chari, R. V., Antibodyconjugate therapeutics: challenges and potential, Clin. Cancer Res.,17(20):6389-97, 2011, Firer, M. A., and Gellerman, G. J., Targeted drugdelivery for cancer therapy: the other side of antibodies, J. Hematol.Oncol., 5:70, 2012, Vlachakis, D. and Kossida, S., Antibody DrugConjugate bioinformatics: drug delivery through the letterbox, Comput.Math. Methods Med., 2013; 2013:282398, Epub 2013 Jun. 19, Lambert, J.M., Drug-conjugated antibodies for the treatment of cancer, Br. J. Clin.Pharmacol., 76(2):248-62, 2013, Concalves, A., Tredan, O., Villanueva,C. and Dumontet, C., Antibody-drug conjugates in oncology: from theconcept to trastuzumab emtansine (T-DM1), Bull. Cancer,99(12):1183-1191, 2012, Newland, A. M., Brentuximab vedotin: aCD-30-directed antibody-cytotoxic drug conjugate, Pharmacotherapy,33(1):93-104, 2013, Lopus, M., Antibody-DM1 conjugates as cancertherapeutics, Cancer Lett., 307(2):113-118, 2011, Chu, Y. W. and Poison,A., Antibody-drug conjugates for the treatment of B-cell non-Hodgkin'slymphoma and leukemia, Future Oncol., 9(3):355-368, 2013, Bertholjotti,I., Antibody-drug conjugate—a new age for personalized cancer treatment,Chimia, 65(9): 746-748, 2011, Vincent, K. J., and Zurini, M., Currentstrategies in antibody engineering: Fc engineering and pH-dependentantigen binding, bispecific antibodies and antibody drug conjugates,Biotechnol. J., 7(12):1444-1450, 2012, Haeuw, J. F., Caussanel, V., andBeck, A., Immunoconjugates, drug-armed antibodies to fight againstcancer, Med. Sci., 25(12):1046-1052, 2009 and Govindan, S. V., andGoldenberg, D. M., Designing immunoconjugates for cancer therapy, ExpertOpin. Biol. Ther., 12(7):873-890, 2012.

Pharmaceutical Compositions and Dosage Forms

The CDK4/6 inhibitor and topoisomerase I inhibitor described herein, orits salt, isotopic analog, or prodrug can be administered in aneffective amount to the host using any suitable approach which achievesthe desired therapeutic result. The amount and timing of active compoundadministered will, of course, be dependent on the host being treated,the instructions of the supervising medical specialist, on the timecourse of the exposure, on the manner of administration, on thepharmacokinetic properties of the particular active compound, and on thejudgment of the prescribing physician. Thus, because of host to hostvariability, the dosages given below are a guideline and the physiciancan titrate doses of the compound to achieve the treatment that thephysician considers appropriate for the host. In considering the degreeof treatment desired, the physician can balance a variety of factorssuch as age and weight of the host, presence of preexisting disease, aswell as presence of other diseases. Pharmaceutical formulations can beprepared for any desired route of administration including, but notlimited to, oral, intravenous, or aerosol administration, as discussedin greater detail below.

The therapeutically effective dosage of any active compound describedherein will be determined by the health care practitioner depending onthe condition, size and age of the patient as well as the route ofdelivery. In one non-limited embodiment, a dosage from about 0.1 toabout 200 mg/kg has therapeutic efficacy, with all weights beingcalculated based upon the weight of the active compound, including thecases where a salt is employed. In some embodiments, the dosage can bethe amount of compound needed to provide a serum concentration of theactive compound of up to between about 1 and 5, 10, 20, 30, or 40 μM. Insome embodiments, a dosage from about 10 mg/kg to about 50 mg/kg can beemployed for oral administration. Typically, a dosage from about 0.5mg/kg to 5 mg/kg can be employed for intramuscular injection. In someembodiments, dosages can be from about 1 μmol/kg to about 50 μmol/kg,or, optionally, between about 22 μmol/kg and about 33 μmol/kg of thecompound for intravenous or oral administration. An oral dosage form caninclude any appropriate amount of active material, including for examplefrom 5 mg to, 50, 100, 200, or 500 mg per tablet or other solid dosageform.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid or as a liquid, or can be administered intramuscularly,intravenously, or by inhalation as a solution, suspension, or emulsion.In some embodiments, the compounds or salts also can be administered byinhalation, intravenously, or intramuscularly as a liposomal suspension.When administered through inhalation the active compound or salt can bein the form of a plurality of solid particles or droplets having anydesired particle size, and for example, from about 0.01, 0.1 or 0.5 toabout 5, 10, 20 or more microns, and optionally from about 1 to about 2microns. Compounds as disclosed in the present invention havedemonstrated good pharmacokinetic and pharmacodynamics properties, forinstance when administered by the oral or intravenous routes.

The pharmaceutical formulations can comprise a CDK4/6 inhibitor and/ortopoisomerase I inhibitor described herein or a pharmaceuticallyacceptable salt thereof, in any pharmaceutically acceptable carrier. Ifa solution is desired, water may be the carrier of choice forwater-soluble compounds or salts. With respect to the water-solublecompounds or salts, an organic vehicle, such as glycerol, propyleneglycol, polyethylene glycol, or mixtures thereof, can be suitable. Inthe latter instance, the organic vehicle can contain a substantialamount of water. The solution in either instance can then be sterilizedin a suitable manner known to those in the art, and for illustration byfiltration through a 0.22-micron filter. Subsequent to sterilization,the solution can be dispensed into appropriate receptacles, such asdepyrogenated glass vials. The dispensing is optionally done by anaseptic method. Sterilized closures can then be placed on the vials and,if desired, the vial contents can be lyophilized.

In addition to the active compounds or their salts, the pharmaceuticalformulations can contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the formulations can contain antimicrobialpreservatives. Useful antimicrobial preservatives include methylparaben,propylparaben, and benzyl alcohol. An antimicrobial preservative istypically employed when the formulations is placed in a vial designedfor multi-dose use. The pharmaceutical formulations described herein canbe lyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate may be employed along withvarious disintegrants such as starch (e.g., potato or tapioca starch)and certain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate, and talc are often very useful for tableting purposes.Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules. Materials in this connection alsoinclude lactose or milk sugar as well as high molecular weightpolyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the compounds of the presentlydisclosed host matter can be combined with various sweetening agents,flavoring agents, coloring agents, emulsifying agents and/or suspendingagents, as well as such diluents as water, ethanol, propylene glycol,glycerin and various like combinations thereof.

In yet another embodiment of the host matter described herein, there areprovided injectable, stable, sterile formulations comprising an activecompound as described herein, or a salt thereof, in a unit dosage formin a sealed container. The compound or salt is provided in the form of alyophilizate, which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form liquid formulation suitablefor injection thereof into a host. When the compound or salt issubstantially water-insoluble, a sufficient amount of emulsifying agent,which is physiologically acceptable, can be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier.Particularly useful emulsifying agents include phosphatidyl cholines andlecithin.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a desired compound described herein or asalt thereof, or a plurality of solid particles of the compound or salt.The desired formulations can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles may for example have a particle size in the range of about 0.5to about 10 microns, and optionally from about 0.5 to about 5 microns.In one embodiment, the solid particles provide for controlled releasethrough the use of a degradable polymer. The solid particles can beobtained by processing the solid compound or a salt thereof, in anyappropriate manner known in the art, such as by micronization.Optionally, the size of the solid particles or droplets can be fromabout 1 to about 2 microns. In this respect, commercial nebulizers areavailable to achieve this purpose. The compounds can be administered viaan aerosol suspension of respirable particles in a manner set forth inU.S. Pat. No. 5,628,984, the disclosure of which is incorporated hereinby reference in its entirety.

Pharmaceutical formulations also are provided which provide a controlledrelease of a compound described herein, including through the use of adegradable polymer, as known in the art.

When the pharmaceutical formulations suitable for administration as anaerosol is in the form of a liquid, the formulations can comprise awater-soluble active compound in a carrier that comprises water. Asurfactant can be present, which lowers the surface tension of theformulations sufficiently to result in the formation of droplets withinthe desired size range when hosted to nebulization.

The term “pharmaceutically acceptable salts” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with hosts (e.g., human hosts) without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe presently disclosed host matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of the presently disclosed compounds. Thesesalts can be prepared during the final isolation and purification of thecompounds or by separately reacting the purified compound in its freebase form with a suitable organic or inorganic acid and isolating thesalt thus formed. Basic compounds are capable of forming a wide varietyof different salts with various inorganic and organic acids. Acidaddition salts of the basic compounds are prepared by contacting thefree base form with a sufficient amount of the desired acid to producethe salt in the conventional manner. The free base form can beregenerated by contacting the salt form with a base and isolating thefree base in the conventional manner. The free base forms may differfrom their respective salt forms in certain physical properties such assolubility in polar solvents. Pharmaceutically acceptable base additionsalts may be formed with metals or amines, such as alkali and alkalineearth metal hydroxides, or of organic amines. Examples of metals used ascations, include, but are not limited to, sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines include, but are notlimited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, and procaine. Thebase addition salts of acidic compounds are prepared by contacting thefree acid form with a sufficient amount of the desired base to producethe salt in the conventional manner. The free acid form can beregenerated by contacting the salt form with an acid and isolating thefree acid in a conventional manner. The free acid forms may differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents.

Salts can be prepared from inorganic acids sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, laurylsulphonate and isethionate salts,and the like. Salts can also be prepared from organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. and the like. Representativesalts include acetate, propionate, caprylate, isobutyrate, oxalate,malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Pharmaceuticallyacceptable salts can include cations based on the alkali and alkalineearth metals, such as sodium, lithium, potassium, calcium, magnesium andthe like, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Also contemplated are the saltsof amino acids such as arginate, gluconate, galacturonate, and the like.See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which isincorporated herein by reference.

Preparation of CDK4/6 Inhibitors

Syntheses

The disclosed CDK4/6 inhibitors can be made by the following generalschemes:

In Scheme 1, Ref-1 is WO 2010/020675 A1; Ref-2 is White, J. D.; et al.J. Org. Chem. 1995, 60, 3600; and Ref-3 Presser, A. and Hufner, AMonatshefte für Chemie 2004, 135, 1015.

In Scheme 2, Ref-1 is WO 2010/020675 A1; Ref-4 is WO 2005/040166 A1; andRef-5 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

In Scheme 3, Ref-1 is WO 2010/020675 A1.

In Scheme 8, Ref-1 is WO 2010/020675 A1; Ref-2 is WO 2005/040166 A1; andRef-3 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

Alternatively, the lactam can be generated by reacting the carboxylicacid with a protected amine in the presence of a strong acid and adehydrating agent, which can be together in one moiety as a strong acidanhydride. Examples of strong acid anhydrides include, but are notlimited to, trifluoroacetic acid anhydride, tribromoacetic acidanhydride, trichloroacetic acid anhydride, or mixed anhydrides. Thedehydrating agent can be a carbodiimide based compound such as but notlimited to DCC (N,N-dicyclohexylcarbodiimide), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC(N,N-diisopropylcarbodiimide). An additional step may be necessary totake off the N-protecting group and the methodologies are known to thoseskilled in the art.

Alternatively, the halogen moiety bonded to the pyrimidine ring can besubstituted with any leaving group that can be displaced by a primaryamine, for example to create an intermediate for a final product such asBr, I, F, SMe, SO₂Me, SOalkyl, SO₂alkyl. See, for ExamplePCT/US2013/037878 to Tavares.

Other amine intermediates and final amine compounds can be synthesizedby those skilled in the art. It will be appreciated that the chemistrycan employ reagents that comprise reactive functionalities that can beprotected and de-protected and will be known to those skilled in the artat the time of the invention. See for example, Greene, T. W. and Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons.

CDK4/6 Inhibitors of the present invention can be synthesized accordingto the generalized Scheme 9. Specific synthesis and characterization ofthe Substituted 2-aminopyrmidines can be found in, for instance,WO2012/061156.

Compounds T, Q, GG, and U were prepared as above and were characterizedby mass spectrometry and NMR as shown below:

Compound T

1H NMR (600 MHz, DMSO-d6) □ ppm 1.47 (br. s., 6H) 1.72 (br. s., 2H) 1.92(br. s., 2H) 2.77 (br. s., 3H) 3.18 (br. s., 2H) 3.46 (br. s., 2H) 3.63(br. s., 2H) 3.66 (d, J=6.15 Hz, 2H) 3.80 (br. s., 2H) 7.25 (s, 1H) 7.63(br. s., 2H) 7.94 (br. s., 1H) 8.10 (br. s., 1H) 8.39 (br. s., 1H) 9.08(br. s., 1H) 11.59 (br. s., 1H). LCMS ESI (M+H) 447.

Compound Q

1H NMR (600 MHz, DMSO-d6) □ ppm 0.82 (d, J=7.32 Hz, 2H) 1.08-1.37 (m,3H) 1.38-1.64 (m, 2H) 1.71 (br. s., 1H) 1.91 (br. s., 1H) 2.80 (br. s.,1H) 3.12 (s, 1H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 4.09 (br. s., 1H)7.26 (s, 1H) 7.52-7.74 (m, 2H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40(br. s., 1H) 9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H).LCMS ESI (M+H) 433.

Compound GG

1H NMR (600 MHz, DMSO-d6) □ ppm 0.85 (br. s., 1H) 1.17-1.39 (m, 7H)1.42-1.58 (m, 2H) 1.67-1.84 (m, 3H) 1.88-2.02 (m, 1H) 2.76-2.93 (m, 1H)3.07-3.22 (m, 1H) 3.29-3.39 (m, 1H) 3.41-3.61 (m, 4H) 3.62-3.76 (m, 4H)3.78-3.88 (m, 1H) 4.12 (br. s., 1H) 7.28 (s, 1H) 7.60-7.76 (m, 2H) 7.98(s, 1H) 8.13 (br. s., 1H) 8.41 (s, 1H) 9.10 (br. s., 1H) 11.21 (br. s.,1H) 11.54 (s, 1H). LCMS ESI (M+H) 475.

Compound U

1H NMR (600 MHz, DMSO-d6) □ ppm 0.84 (t, J=7.61 Hz, 2H) 1.13-1.39 (m,4H) 1.46 (d, J=14.05 Hz, 2H) 1.64-1.99 (m, 6H) 2.21 (br. s., 1H)2.66-2.89 (m, 2H) 3.06 (br. s., 1H) 3.24-3.36 (m, 1H) 3.37-3.50 (m, 2H)3.56-3.72 (m, 2H) 3.77-4.00 (m, 4H) 4.02-4.19 (m, 2H) 7.25 (s, 1H)7.50-7.75 (m, 2H) 7.89 (d, J=2.93 Hz, 1H) 8.14 (d, J=7.32 Hz, 1H) 8.38(br. s., 1H) 9.06 (s, 1H) 11.53 (br. s., 1H). LCMS ESI (M+H) 517.

EXAMPLES

Intermediates B, E, K, L, 1A, 1F and 1CA were synthesized according toU.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X. andStrum, J. C.

The patents WO 2013/148748 entitled Lactam Kinase Inhibitors to Tavares,F. X., WO 2013/163239 entitled Synthesis of Lactams to Tavares, F. X.,and U.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X.and Strum, J. C. are incorporated by reference herein in their entirety.

Example 1 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4yl)amino]ethyl]carbamate, Compound 1

To a solution of 5-bromo-2,4-dichloropyrimidine (3.2 g, 0.0135 mol) inethanol (80 mL) was added Hunig's base (3.0 mL) followed by the additionof a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (2.5 g,0.0156 mole) in ethanol (20 mL). The contents were stirred overnight for20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (200 mL)and water (100 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. ¹HNMR(d6-DMSO) δ ppm 8.21 (s, 1H), 7.62 (brs, 1H), 7.27 (brs, 1H), 3.39 (m,2H), 3.12 (m, 2H), 1.34 (s, 9H). LCMS (ESI) 351 (M+H).

Example 2 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate,Compound 2

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (1.265 g,3.6 mmol) in THF (10 mL) was added the acetal (0.778 mL, 5.43 mmol),Pd(dppf)CH₂Cl₂ (148 mg), and triethylamine (0.757 mL, 5.43 mmol). Thecontents were degassed and then purged with nitrogen. To this was thenadded CuI (29 mg). The reaction mixture was heated at reflux for 48 hrs.After cooling, the contents were filtered over CELITE™ and concentrated.Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹HNMR (d6-DMSO) δ ppm 8.18 (s, 1H), 7.63 (brs, 1H), 7.40 (brs, 1H), 5.55(s, 1H), 3.70 (m, 2H), 3.60 (m, 2H), 3.42 (m, 2H), 3.15 (m, 2H),1.19-1.16 (m, 15H). LCMS (ESI) 399 (M+H).

Example 3 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 3

To a solution of the coupled product (2.1 g, 0.00526 mole) in THF (30mL) was added TBAF solid (7.0 g). The contents were heated to andmaintained at 65 degrees for 2 hrs. Concentration followed by columnchromatography using ethyl acetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d6-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 4 Synthesis of tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate,Compound 4

To the acetal (900 mg) from the preceeding step was added AcOH (8.0 mL)and water (1.0 mL). The reaction was stirred at room temperature for 16hrs. Conc. and column chromatography over silica gel using ethylacetate/hexanes (0-60%) afforded tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate asa foam (0.510 g). ¹HNMR (d6-DMSO) δ ppm 9.98 (s, 1H), 9.18 (s, 1H), 7.66(s, 1H), 6.80 (brs, 1H), 4.52 (m, 2H), 4.36 (m, 2H), 1.14 (s, 9H). LCMS(ESI) 325 (M+H).

Example 5 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 5

To the aldehyde (0.940 g) from the preceeding step in DMF (4 mL) wasadded oxone (1.95 g, 1.1 eq). The contents were stirred at room temp for7 hrs. Silica gel column chromatography using hexane/ethyl acetate(0-100%) afforded7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g). ¹HNMR (d6-DMSO) δ ppm 9.11 (s, 1H), 7.39 (s, 1H), 4.38(m, 2H), 4.15 (m, 2H), 1.48 (m, 9H). LCMS (ESI) 341 (M+H).

Example 6 Synthesis of methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate,Compound 6

To a solution of 2-chloro-7-propyl-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g, 0.00156 mole) from the preceeding step in toluene (3.5mL) and MeOH (1 mL) was added TMS-diazomethane (1.2 mL). After stirringovernight at room temperature, the excess of TMS-diazomethane wasquenched with acetic acid (3 mL) and the reaction was concentrated undervacuum. The residue was purified by silica gel column chromatographywith hexane/ethyl acetate (0-70%) to afford methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylateas an off white solid (0.52 g). ¹HNMR (d6-DMSO) δ ppm 9.10 (s, 1H), 7.45(s, 1H), 6.81 (brs, 1H) 4.60 (m, 2H), 3.91 (s, 3H), 3.29 (m, 2H), 1.18(m, 9H) LCMS (ESI) 355 (M+H).

Example 7 Synthesis of Chloro Tricyclic Amide, Compound 7

To methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate(0.50 g, 0.0014 mole) from the preceeding step in dichloromethane (2.0mL) was added TFA (0.830 mL). The contents were stirred at roomtemperature for 1 hr. Concentration under vacuum afforded the crudeamino ester which was suspended in toluene (5 mL) and Hunig's base (0.5mL). The contents were heated at reflux for 2 hrs. Concentrationfollowed by silica gel column chromatography using hexane/ethyl acetate(0-50%) afforded the desired chloro tricyclic amide (0.260 g). ¹HNMR(d6-DMSO) δ ppm 9.08 (s, 1H), 8.48 (brs, 1H), 7.21 (s, 1H) 4.33 (m, 2H),3.64 (m, 2H). LCMS (ESI) 223 (M+H).

Example 8 Synthesis of chloro-N-methyltricyclic amide, Compound 8

To a solution of the chloro tricycliclactam, Compound 7, (185 mg,0.00083 mole) in DMF (2.0 mL) was added sodium hydride (55% dispersionin oil, 52 mg). After stirring for 15 mins, methyl iodide (62 μL, 1.2eq). The contents were stirred at room temperature for 30 mins. Afterthe addition of methanol (5 mL), sat NaHCO₃ was added followed by theaddition of ethyl acetate. Separation of the organic layer followed bydrying with magnesium sulfate and concentration under vacuum affordedthe N-methylated amide in quantitative yield. ¹HNMR (d6-DMSO) δ ppm 9.05(s, 1H), 7.17 (s, 1H) 4.38 (m, 2H), 3.80 (m, 2H), 3.05 (s, 3H). LCMS(ESI) 237 (M+H).

Example 9 Synthesis of 1-methyl-4-(6-nitro-3-pyridyl)piperazine,Compound 9

To 5-bromo-2-nitropyridine (4.93 g, 24.3 mmole) in DMF (20 mL) was addedN-methylpiperazine (2.96 g, 1.1 eq) followed by the addition of DIPEA(4.65 mL, 26.7 mmole). The contents were heated at 90 degrees for 24hrs. After addition of ethyl acetate (200 mL), water (100 mL) was addedand the layers separated. Drying followed by concentration afforded thecrude product which was purified by silica gel column chromatographyusing (0-10%) DCM/Methanol. ¹HNMR (d6-DMSO) δ ppm 8.26 (s, 1H), 8.15(1H, d, J=9.3 Hz), 7.49 (1H, d, J=9.4 Hz), 3.50 (m, 4H), 2.49 (m, 4H),2.22 (s, 3H).

Example 10 Synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine,Compound 10

To 1-methyl-4-(6-nitro-3-pyridyl)piperazine (3.4 g) in ethyl acetate(100 mL) and ethanol (100 mL) was added 10% Pd/C (400 mg) and then thereaction was stirred under hydrogen (10 psi) overnight. After filtrationthrough CELITE™, the solvents were evaporated and the crude product waspurified by silica gel column chromatography using DCM/7N ammonia inMeOH (0-5%) to afford 5-(4-methylpiperazin-1-yl)pyridin-2-amine (2.2 g).¹HNMR (d6-DMSO) δ ppm 7.56 (1H, d, J=3 Hz), 7.13 (1H, m), 6.36 (1H, d,J=8.8 Hz), 5.33 (brs, 2H), 2.88 (m, 4H), 2.47 (m, 4H), 2.16 (s, 3H).

Example 11 Synthesis of tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate, Compound 11

This compound was prepared as described in WO 2010/020675 A1.

Example 12 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound 12

To benzyl N-[1-(hydroxymethyl)-2-methyl-propyl]carbamate (11.0 g, 0.0464mole) in dioxane (100 mL) cooled to 0° C. was added diphenylphosphorylazide (10.99 mL, 1.1 eq) followed by the addition of DBU (8.32 mL, 1.2eq). The contents were allowed to warm to room temperature and stirredfor 16 hrs. After the addition of ethyl acetate (300 mL) and water (100mL), the organic layer was separated and washed with satd. NaHCO₃ (100mL). The organic layer was then dried (magnesium sulfate) andconcentrated under vacuum. To this intermediate in DMSO (100 mL) wasadded sodium azide (7.54 g) and the contents then heated to 90 degreesfor 2 hrs. After addition of ethyl acetate and water the layers wereseparated. The organic layer was dried with magnesium sulfate followedby concentration under vacuum to afford an oil that was purified bysilica gel column chromatography using hexane/ethyl acetate (0-70%) toafford benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate 6.9 g as acolorless oil.

To benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate (6.9 g, 0.0263mole) in THF (100 mL) was added triphenyl phosphine (7.59 g, 1.1 eq).The contents were stirred for 20 hrs. After addition of water (10 mL),and stirring for an additional 6 hrs, ethyl acetate was added and thelayers separated. After drying with magnesium sulfate and concentrationunder vacuum, the crude product was purified by silica gel columnchromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-2-methyl-propyl] carbamate as a yellow oil.

To benzyl N-[1-(aminomethyl)-2-methyl-propyl] carbamate (4.65 g, 0.019mole) in THF (70 mL) was added 2N NaOH (20 mL) followed by the additionof di-tert-butyl dicarbonate (5.15 g, 1.2 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product waspurified using hexane/ethyl acetate (0-40%) over a silica gel column toafford intermediate A, tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, (6.1 g). ¹HNMR(600 MHz, CHLOROFORM-d) δ ppm 0.89 (d, J=6.73 Hz, 3H) 0.92 (d, J=6.73Hz, 3H) 1.38 (s, 9H) 1.70-1.81 (m, 1H) 3.18 (d, J=5.56 Hz, 2H) 3.47-3.60(m, 1H) 4.76 (s, 1H) 4.89 (d, J=7.90 Hz, 1H) 5.07 (s, 2H) 7.25-7.36 (m,5H). LCMS (ESI) 337 (M+H).

Example 13 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound 13

To a solution of benzyl N-[1-(hydroxymethyl)-3-methyl-butyl]carbamate(6.3 g, 0.025 mole) in DCM (100 mL) was added diisopropylethyl amine(5.25 mL, 1.2 eq) followed by the addition of methane sulfonylchloride(2.13 mL, 1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL)was added and the organic layer separated. After drying with magnesiumsulfate and concentration under vacuum, the crude[2-(benzyloxycarbonylamino)-4-methyl-pentyl]methanesulfonate which wastaken directly to the next step.

To the crude [2-(benzyloxycarbonylamino)-4-methyl-pentyl]methanesulfonate from the above reaction in DMF (50 mL), was addedsodium azide 2.43 g. The reaction mixture was then heated to 85 degreesfor 3 hrs. After cooling, ethyl acetate (300 mL) and water was added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THF (100 mL) followed by triphenylphosphine 7.21 g and stirredunder nitrogen for 16 hrs. After addition of water (10 mL), and stirringfor an additional 6 hrs, ethyl acetate was added and the layersseparated. After drying with magnesium sulfate and concentration undervacuum, the crude product was columned using DCM/MeOH (0-10%) to affordbenzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g).

To benzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g, 0.018mole) in THF (60 mL) was added 2N NaOH (18 mL) followed by the additionof di-tert-butyl dicarbonate (4.19 g, 1.07 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product wastaken to the next step. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.89 (d,J=6.73 Hz, 6H) 1.25-1.34 (m, 1H) 1.39 (s, 9H) 1.57-1.71 (m, 2H)3.04-3.26 (m, 2H) 3.68-3.80 (m, 1H) 4.72-4.89 (m, 2H) 5.06 (s, 2H)7.25-7.38 (m, 5H). LCMS (ESI) 351 (M+H).

Example 14 Synthesis of tert-butylN-[(2R)-2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound14

Compound 14 was synthesized from benzylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl]carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 15 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-butyl]carbamate, Compound 15

Compound 15 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl]carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 16 Synthesis of tert-butylN-[(1S)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 16

To a solution of tert-butylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl]carbamate carbamate (6.3 g,0.025 mole) in THF (100 mL) was added diisopropylethyl amine (5.25 mL,1.2 eq) followed by the addition of methane sulfonylchloride (2.13 mL,1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL) was addedand the organic layer separated. After drying with magnesium sulfate andconcentration under vacuum, the crude[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl] methanesulfonate wastaken directly to the next step.

To the crude [(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]methanesulfonate from the above reaction in DMSO (50 mL), was addedsodium azide (2.43 g). The reaction mixture was then heated to 85degrees for 3 hrs. After cooling, ethyl acetate (300 mL) and water wereadded. The organic layer was separated, dried with magnesium sulfate andthen concentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THF (100 mL) followed by triphenylphosphine (7.21 g) and thereation was stirred under nitrogen for 16 hrs. After addition of water(10 mL), and stirring for an additional 6 hrs, ethyl acetate was addedand the layers separated. After drying with magnesium sulfate andconcentration under vacuum, the crude product was purified by silica gelcolumn chromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g). LCMS (ESI) 203(M+H).

Example 17 Synthesis of tert-butylN-[(1R)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 17

Compound 17 was synthesized from tert-butylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl] carbamate using a similarsynthetic sequence as described for Compound 16. The analytical data(NMR and mass spec) was consistent with Compound 16.

Example 18 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound18

Compound 18 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-3-methyl-butyl]carbamate using a similarsynthetic sequence as described for Compound 13. The analytical data(NMR and mass spec) was consistent with Compound 13.

Example 19 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-2-phenyl-ethyl] carbamate, Compound19

Compound 19 was synthesized from benzylN-[(1S)-2-hydroxy-1-phenyl-ethyl] carbamate using a similar syntheticsequence as described for Compound 13. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.20-1.33 (m, 9H) 3.11 (t, J=6.29 Hz, 2H) 4.59-4.68 (m, 1H) 4.88-5.01(m, 2H) 6.81 (t, J=5.42 Hz, 1H) 7.14-7.35 (m, 10H) 7.69 (d, J=8.49 Hz,1H). LCMS (ESI) 371 (M+H).

Example 20 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-pentyl] carbamate, Compound20

Compound 20 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-butyl]carbamate using a similarsynthetic sequence as described for Compound 13. ¹HNMR (600 MHz,CHLOROFORM-d) δ ppm 0.85-0.92 (m, 6H) 1.05-1.15 (m, 1H) 1.35-1.41 (m,9H) 1.45-1.56 (m, 2H) 3.14-3.24 (m, 2H) 3.54-3.64 (m, 1H) 4.78 (s, 1H)4.96 (d, J=7.91 Hz, 1H) 5.06 (s, 2H) 7.27-7.37 (m, 5H). LCMS (ESI) 351(M+H).

Example 21 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3,3-dimethyl-butyl] carbamate,Compound 21

Compound 21 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2,2-dimethyl-propyl]carbamate using a similarsynthetic sequence as described for Compound 13. LCMS (ESI) 351.

Example 22 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl] carbamate, Compound 22

To a solution of benzyl N-[1-(aminomethyl)cyclohexyl]carbamate (10.0 g,0.0381 mole) in THF (150 mL) was added di-tert-butyl dicarbonate (9.15g, 1.1 eq) and the contents were stirred at room temperature for 16 hrs.Ethyl acetate and water were then added. The organic layer wasseparated, dried over magnesium sulfate and then concentrated undervacuum to afford tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl] carbamate (13.1 g).¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.92-1.54 (m, 17H) 1.76-2.06 (m, 2H) 3.09(d, J=6.15 Hz, 2H) 4.92 (s, 2H) 6.63 (d, J=17.27 Hz, 1H) 7.16-7.49 (m,6H). LCMS (ESI) 363 (M+H).

Example 23 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl] carbamate, Compound 23

tert-butyl N-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl]carbamatewas synthesized in an analogous manner to tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl]carbamate. LCMS (ESI)349 (M+H).

Example 24 Synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine,Compound 24

To 5-bromo-2-nitropyridine (1.2 g, 5.9 mmol) in DMSO (4 mL) was added1-(4-piperidyl)piperidine (1.0 g, 5.9 mmole) and triethylamine (0.99 mL,7.1 mmole). The contents were heated to 120° C. in a CEM Discoverymicrowave system for 3 hours. The crude reaction was then purified bysilica gel column chromatography with DCM/methanol (0-20%) to afford2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine as an oil (457 mg).¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.26-1.36 (m, 2H) 1.43 (m, 6H) 1.76 (m,2H) 2.37 (m, 5H) 2.94 (t, J=12.74 Hz, 2H) 4.06 (d, J=13.47 Hz, 2H) 7.41(dd, J=9.37, 2.64 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.64 Hz,1H).

Example 25 Synthesis of 5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine,Compound 25

5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.13-1.37 (m, 6H) 1.40-1.63 (m, 6H) 1.71 (m, 2H), 2.24 (m, 1H) 2.43(m, 2H) 3.33 (d, J=12.30 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=8.78 Hz, 1H)7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS (ESI) 261(M+H).

Example 26 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine, Compound 26

4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.41 (m, 2H) 1.82 (m, 2H) 2.42 (m, 5H) 2.98 (t, J=12.44Hz, 2H) 3.52 (s, 4H) 4.04 (d, J=12.88 Hz, 2H) 7.42 (d, J=9.37 Hz, 1H)8.08 (d, J=9.08 Hz, 1H) 8.21 (s, 1H).

Example 27 Synthesis of 5-(4-morpholino-1-piperidyl) pyridin-2-amine,Compound 27

5-(4-morpholino-1-piperidyl)pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.34-1.52 (m, 2H) 1.78 (m, 2H) 2.14 (m, 1H) 2.43 (m, 4H) 3.32 (d,J=12.30 Hz, 4H) 3.47-3.59 (m, 4H) 5.32 (s, 2H) 6.34 (d, J=8.78 Hz, 1H)7.11 (dd, J=8.93, 2.78 Hz, 1H) 7.47-7.62 (m, 1H). LCMS (ESI) 263 (M+H).

Example 28 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]thiomorpholine, Compound 28

4-[1-(6-nitro-3-pyridyl)-4-piperidyl] thiomorpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.40-1.52 (m, 2H) 1.71 (m, 2H) 2.49-2.55 (m, 4H)2.56-2.63 (m, 1H) 2.68-2.75 (m, 4H) 2.88-2.98 (m, 2H) 4.09 (d, J=13.18Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d,J=3.22 Hz, 1H).

Example 29 Synthesis of 5-(4-thiomorpholino-1-piperidyl)pyridin-2-amine, Compound 29

5-(4-thiomorpholino-1-piperidyl) pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.47-1.59 (m, 2H) 1.65 (m, 2H) 2.22-2.38 (m, 1H) 2.50-2.59 (m, 6H)2.68-2.82 (m, 4H) 3.33 (d, J=12.00 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=9.08Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS(ESI) 279 (M+H).

Example 30 Synthesis of 2-nitro-5-(1-piperidyl)pyridine, Compound 30

2-nitro-5-(1-piperidyl) pyridine was synthesized in a manner similar tothat used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.56 (m, 6H) 3.49 (d, J=4.39 Hz, 4H) 7.30-7.47 (m, 1H)8.02-8.12 (m, 1H) 8.15-8.26 (m, 1H).

Example 31 Synthesis of 5-(1-piperidyl)pyridin-2-amine, Compound 31

5-(1-piperidyl) pyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.39-1.46 (m, 2H) 1.51-1.62 (m, 4H)2.75-2.92 (m, 4H) 5.30 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.09 (dd, J=8.78,2.93 Hz, 1H) 7.54 (d, J=2.93 Hz, 1H). LCMS (ESI) 178 (M+H).

Example 32 Synthesis of 4-(6-nitro-3-pyridyl) thiomorpholine, Compound32

4-(6-nitro-3-pyridyl) thiomorpholine was synthesized in a manner similarto that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 2.56-2.69 (m, 4H) 3.79-3.92 (m, 4H) 7.43 (dd, J=9.22,3.07 Hz, 1H) 8.10 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.93 Hz, 1H).

Example 33 Synthesis of 5-thiomorpholinopyridin-2-amine, Compound 33

5-thiomorpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 2.59-2.73 (m, 4H) 3.04-3.20 (m, 4H) 5.41(s, 2H) 6.35 (d, J=8.78 Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.57 (d,J=2.64 Hz, 1H). LCMS (ESI) 196 (M+H).

Example 34 Synthesis of tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 34

tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas synthesized in a manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.33 (d, J=32.21 Hz, 11H) 1.91 (m, 2H) 3.15 (d, J=10.25Hz, 1H) 3.58 (m, 1H) 4.46 (m, 1H) 4.83 (s, 1H) 7.16 (s, 1H) 7.94 (s, 1H)8.05-8.16 (m, 1H).

Example 35 Synthesis of tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 35

tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas prepared in a manner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.31 (d, J=31.91 Hz, 11H) 1.83 (m, 2H) 2.71-2.82 (m, 1H) 3.44 (m,1H) 4.30 (d, 2H) 5.08 (s, 2H) 6.35 (d, J=8.78 Hz, 1H) 6.77-6.91 (m, 1H)7.33 (s, 1H). LCMS (ESI) 291 (M+H).

Example 36 Synthesis of N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine, Compound 36

N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30-1.45 (m, 2H) 1.79 (m, 2H) 2.14 (s, 6H) 2.33 (m, 1H)2.92-3.04 (m, 2H) 4.03 (d, J=13.76 Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz,1H) 8.04-8.11 (m, 1H) 8.21 (d, J=2.93 Hz, 1H).

Example 37 Synthesis of 5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine, Compound 37

5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.35-1.50 (m, 2H) 1.69-1.81 (m, 2H) 2.00-2.10 (m, 1H) 2.11-2.22 (s,6H) 3.17-3.36 (m, 4H) 5.19-5.38 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.10(dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.63 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 38 Synthesis of 4-(6-nitro-3-pyridyl) morpholine, Compound 38

4-(6-nitro-3-pyridyl) morpholine was synthesized in a manner similar tothat used in the synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine.

Example 39 Synthesis of 5-morpholinopyridin-2-amine, Compound 39

5-morpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 2.91-3.00 (m, 4H) 3.76-3.84 (m, 4H)4.19 (br. s., 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78, 2.93 Hz, 1H)7.72 (d, J=2.93 Hz, 1H).

Example 40 Synthesis of 5-(4-isobutylpiperazin-1-yl) pyridin-2-amine,Compound 40

1-isobutyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then converted5-(4-isobutylpiperazin-1-yl)pyridin-2-amine in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88 (d, J=6.73 Hz, 6H) 1.71-1.84(m, 1H) 2.10 (d, J=7.32 Hz, 2H) 2.46-2.58 (m, 4H) 2.97-3.07 (m, 4H) 4.12(s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.75 (d,J=2.93 Hz, 1H). LCMS (ESI) 235 (M+H).

Example 41 Synthesis of 5-(4-isopropylpiperazin-1-yl) pyridin-2-amine,Compound 41

1-isopropyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-(4-isopropylpiperazin-1-yl)pyridin-2-amine in a manner similar tothat used in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.06 (d, J=6.44 Hz, 6H) 2.59-2.75(m, 5H) 2.97-3.10 (m, 4H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.15 (dd,J=9.08, 2.93 Hz, 1H) 7.76 (d, J=2.93 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 42 Synthesis of5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine, Compound 42

(2S,6R)-2,6-dimethyl-4-(6-nitro-3-pyridyl)morpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.20 (d, J=6.44 Hz, 6H) 2.27-2.39 (m, 2H) 3.11-3.21 (m, 2H)3.70-3.84 (m, 2H) 4.15 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78,2.93 Hz, 1H) 7.72 (d, J=2.63 Hz, 1H). LCMS (ESI) 208 (M+H).

Example 43 Synthesis of5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine, Compound 43

(3S,5R)-3,5-dimethyl-1-(6-nitro-3-pyridyl)piperazine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.09 (d, J=6.44 Hz, 6H) 2.20 (t, J=10.83 Hz, 2H) 2.95-3.08 (m, 2H)3.23 (dd, J=11.71, 2.05 Hz, 2H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H)7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.73 (d, J=2.63 Hz, 1H). LCMS (ESI) 207(M+H).

Example 44 Synthesis of Compound 44

tert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate

A solution of intermediate A in ethanol (100 mL) was hydrogenated under30 psi of hydrogen using 10% Pd/C (0.7 g) in a pressure bomb for 7 hrs.After filtration of the reaction mixture through CELITE™, the organiclayer was concentrated under vacuum to afford tert-butylN-(2-amino-3-methyl-butyl) carbamate (3.8 g).

To a solution of 5-bromo-2,4-dichloro-pyrimidine (7.11 g, 0.0312 mole)in ethanol (100 mL) was added diisopropylethyl amine (5.45 mL, 1.0 eq)and tert-butyl N-(2-amino-3-methyl-butyl) carbamate (6.31 g, 0.0312mole). The reaction mixture was stirred at room temperature for 20 hrs.After concentration under vacuum, ethyl acetate and water were added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to affordtert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77-0.85 (d, J=6.5 Hz, 3H)0.87 (d, J=6.73 Hz, 3H) 1.31-1.39 (m, 9H) 1.82-1.93 (m, 1H) 2.94 (d,J=5.56 Hz, 1H) 3.08-3.22 (m, 2H) 3.98 (d, J=8.20 Hz, 1H) 6.96 (d, J=8.78Hz, 1H) 8.21 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamate

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamatewas synthesized by hosting tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto Sonogoshira conditions as described for tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatefollowed by subsequent treatment with TBAF as described in the synthesisof tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11 (d, J=6.44 Hz, 3H) 1.18 (t, J=7.03Hz, 6H) 1.21-1.26 (m, 12H) 2.88 (br. s., 1H) 3.43-3.78 (m, 6H) 3.97-4.08(m, 1H) 5.61 (s, 1H) 6.65 (s, 1H) 6.71-6.78 (m, 1H) 8.87 (s, 1H). LCMS(ESI) 441 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

To a solution tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatein THF was added TBAF and the contents were heated at reflux for 3 hrs.Ethyl acetate and water were then added and the organic layer separated,dried with magnesium sulfate and then concentrated under vacuum. To thiscrude reaction was added acetic acid/water (9:1) and the contents werestirred for 12 hrs at room temperature. After concentration undervacuum, sat NaHCO₃ and ethyl acetate were added. The organic layer wasseparated, dried and then concentrated under vacuum. The crude reactionproduct thus obtained was dissolved in DMF, oxone was then added and thecontents stirred for 3 hrs. After addition of ethyl acetate, thereaction mixture was filtered through CELITE™ and concentrated undervacuum. Column chromatography of the crude product over silica gel usinghexane/ethyl acetate (0-100%) afforded7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=7.03 Hz, 3H) 0.97 (d,J=6.73 Hz, 3H) 1.52 (s, 9H) 1.99-2.23 (m, 1H) 3.98 (dd, J=14.05, 3.51Hz, 1H) 4.47-4.71 (m, 2H) 7.47 (s, 1H) 9.17 (s, 1H). LCMS (ESI) 383(M+H).

Compound 44

To7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.050 g, 0.00013 mole) in DCM (1.5 mL) was added DIC (32.7 mg) andDMAP (10 mg). The contents were stirred for 2 hrs. Trifluoroacetic acid(0.4 mL) was then added and stirring continued for an additional 30minutes. After addition of satd NaHCO₃ to neutralize the excess acid,ethyl acetate was added and the organic layer separated, dried usingmagnesium sulfate and then concentrated under vacuum. The crude productwas purified by silica gel column chromatography using hexane/ethylacetate (0-100%) to afford the product. ¹HNMR (600 MHz, DMSO-d₆) δ ppm0.72 (d, J=6.73 Hz, 3H) 0.97 (d, J=6.73 Hz, 3H) 2.09-2.22 (m, 1H) 3.57(dd, J=13.18, 4.98 Hz, 1H) 3.72 (dd, J=13.61, 4.25 Hz, 1H) 4.53 (dd,J=8.05, 3.95 Hz, 1H) 7.20 (s, 1H) 8.34 (d, J=4.98 Hz, 1H) 9.08 (s, 1H).LCMS (ESI) 265 (M+H).

Example 45 Synthesis of Compound 45

Compound 14 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2R)-2-amino-3-methyl-butyl] carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 45 Theanalytical data is consistent with that reported for the racemate(Intermediate 1A).

Example 46 Synthesis of Compound 46

Compound 15 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2S)-2-amino-3-methyl-butyl]carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 46. Theanalytical data (NMR and LCMS) was consistent with that reported for theracemate Compound 44.

Example 47 Synthesis of Compound 47

To a solution of Compound 44 (80 mg, 0.00030 mole) in DMF (3 mL) wasadded a 60% dispersion of sodium hydride in oil (40 mg). After stirringfor 15 minutes, methyl iodide (37 μL, 2 eq) was added. The contents werestirred at room temperature for 30 minutes. Saturated NaHCO₃ was thenadded followed by ethyl acetate. The organic layer was dried withmagnesium sulfate and then concentrated under vacuum to afford theproduct. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (d, J=6.73 Hz, 3H) 0.91 (d,J=6.73 Hz, 3H) 2.04-2.20 (m, 1H) 3.04 (s, 3H) 3.69 (dd, J=13.76, 1.17Hz, 1H) 3.96 (dd, J=13.76, 4.68 Hz, 1H) 4.58 (dd, J=7.32, 3.51 Hz, 1H)7.16 (s, 1H) 9.05 (s, 1H). LCMS (ESI) 279 (M+H).

Example 48 Synthesis of Compound 48

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate

Compound 18 was hydrogenated with 10% Pd/C in ethanol under a blanket ofhydrogen at 50 psi in a pressure bomb to afford tert-butylN-[(2S)-2-amino-4-methyl-pentyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.91 (d, J=6.44 Hz, 3H) 0.94 (d,J=6.44 Hz, 3H) 1.32-1.51 (m, 11H) 1.55-1.67 (m, 1H) 3.28 (t, J=5.86 Hz,2H) 4.21-4.42 (m, 1H) 4.84 (s, 1H) 5.84 (d, J=7.32 Hz, 1H) 8.07 (s, 1H).LCMS (ESI) 407 (M+H).

To a solution of tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate(5.0 g, 12.3 mmole) in tolune (36 mL) and triethylamine (7.2 mL) wasadded under nitrogen, 3,3-diethoxyprop-1-yne (2.8 mL, 19.7 mmole),Pd₂(dba)₃ (1.1 g, 1.23 mmole), and triphenylarsine (3.8 g, 12.3 mmole).The contents were heated to 70 degrees for 24 hrs. After cooling to roomtemperature, the reaction mixture was filtered through CELITE™ and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to afford(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 3H) 0.97 (d,J=6.44 Hz, 3H) 1.47 (s, 9H) 1.49-1.54 (m, 1H) 1.56 (t, J=7.17 Hz, 2H)3.98 (dd, J=13.91, 3.07 Hz, 1H) 3.76 (dd, J=13.31, 4.13 Hz, 1H) 4.38 (d,J=14.05 Hz, 1H) 4.90 (t, J=7.17 Hz, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(M+H) 397.

Compound 48 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d,J=6.73 Hz, 3H) 0.97 (d, J=6.44 Hz, 3H) 1.34-1.46 (m, 1H) 1.48-1.65 (m,2H) 3.40 (dd, J=13.32, 5.42 Hz, 1H) 3.76 (dd, J=13.47, 4.10 Hz, 1H)4.76-4.92 (m, 1H) 7.17 (s, 1H) 8.34 (d, J=5.27 Hz, 1H) 9.04 (s, 1H).LCMS (ESI) 279 (M+H).

Example 49 Synthesis of Compound 49

Compound 49 was synthesized in a manner similar to that described forCompound 47. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d, J=6.44 Hz, 3H) 0.97(d, J=6.44 Hz, 3H) 1.37-1.68 (m, 3H) 3.04 (s, 3H) 3.56 (d, J=13.47 Hz,1H) 4.00 (dd, J=13.32, 4.25 Hz, 1H) 4.82-4.94 (m, 1H) 7.16 (s, 1H) 9.03(s, 1H). LCMS (ESI) 293 (M+H).

Example 50 Synthesis of Compound 50

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate

Compound 20 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3-methyl-pentyl]carbamate which was reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88-0.95 (m, 6H) 1.11-1.20 (m, 1H)1.34 (s, 9H) 1.44-1.54 (m, 1H) 1.64-1.72 (m, 1H) 3.17-3.27 (m, 1H)3.33-3.43 (m, 1H) 4.11-4.21 (m, 1H) 4.81 (s, 1H) 5.92 (d, J=8.20 Hz, 1H)8.05 (s, 1H). LCMS (ESI) 407.

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.76-0.89 (m, 6H) 1.03 (q, J=7.22 Hz, 3H)1.10-1.17 (m, 3H) 1.25-1.42 (m, 11H) 1.59-1.73 (m, 1H) 3.35-3.47 (m, 4H)3.51-3.73 (m, 2H) 3.99-4.11 (m, 1H) 5.52-5.56 (m, 1H) 6.76-7.03 (m, 2H)8.12-8.23 (m, 1H). LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.80 (t, J=7.47 Hz, 3H) 0.86 (d,J=7.03 Hz, 3H) 1.06-1.30 (m, 2H) 1.48 (s, 9H) 1.79-1.96 (m, 1H) 3.95(dd, J=14.05, 3.22 Hz, 1H) 4.52 (d, J=14.35 Hz, 1H) 4.61-4.73 (m, 1H)7.43 (s, 1H) 9.13 (s, 1H). LCMS (ESI) 397 (M+H).

Compound 50 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (t,J=7.32 Hz, 3H) 0.89 (d, J=6.73 Hz, 3H) 1.00-1.12 (m, 2H) 1.82-1.94 (m,1H) 3.55 (dd, J=13.91, 4.83 Hz, 1H) 3.70 (dd, J=13.61, 4.25 Hz, 1H) 4.57(dd, J=7.91, 4.10 Hz, 1H) 7.17 (s, 1H) 8.31 (d, J=5.27 Hz, 1H) 9.05 (s,1H). LCMS (ESI) 279 (M+H).

Example 51 Synthesis of Compound 51

Compound 51 was synthesized in a manner similar to Compound 47. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77 (t, J=7.47 Hz, 3H) 0.84 (d, J=6.73 Hz, 3H)1.07-1.16 (m, 2H) 1.82-1.95 (m, 1H) 3.03 (s, 3H) 3.68 (d, J=13.76 Hz,1H) 3.96 (dd, J=13.76, 4.39 Hz, 1H) 4.59-4.70 (m, 1H) 7.16 (s, 1H) 9.04(s, 1H). LCMS (ESI) 293 (M+H).

Example 52 Synthesis of Compound 52

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3,3-dimethyl-butyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate.LCMS (ESI) 407 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 397 (M+H).

Intermediate 1F was synthesized using an analogous synthetic sequence asthat described for intermediate 1A. LCMS (ESI) 279 (M+H).

Example 53 Synthesis of Compound 53

Compound 53 was synthesized in a manner similar to that described forIntermediate 1CA. LCMS (ESI) 293 (M+H).

Example 54 Synthesis of Compound 54

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-2-phenyl-ethyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.32 (s, 9H) 3.29-3.50 (m, 2H) 5.12-5.24(m, 1H) 7.10 (t, J=5.27 Hz, 1H) 7.21 (t, J=6.88 Hz, 1H) 7.26-7.34 (m,4H) 7.89 (d, J=7.32 Hz, 1H) 8.24 (s, 1H). LCMS (ESI) 427 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.14 (t, J=7.03 Hz, 6H) 1.32 (s, 9H) 3.39(s, 2H) 3.52-3.61 (m, 2H) 3.64-3.73 (m, 2H) 5.17-5.26 (m, 1H) 5.57 (s,1H) 7.07-7.14 (m, 1H) 7.20-7.25 (m, 1H) 7.26-7.33 (m, 4H) 7.90 (d,J=7.61 Hz, 1H) 8.19 (s, 1H). LCMS (ESI) 475 (M+H).

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 417 (M+H).

Compound 54

Compound 54 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.58-3.69(m, 1H) 4.13 (dd, J=13.47, 4.39 Hz, 1H) 6.07 (d, J=3.81 Hz, 1H) 6.85 (d,J=7.32 Hz, 2H) 7.19-7.31 (m, 3H) 7.34 (s, 1H) 8.27 (d, J=5.27 Hz, 1H)9.13 (s, 1H). LCMS (ESI) 299 (M+H).

Example 55 Synthesis of Compound 55

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]carbamate

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Eusing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.95-1.02 (m, 6H) 1.35-1.45 (m, 9H)1.75-1.90 (m, 1H) 3.35-3.48 (m, 1H) 3.52-3.61 (m, 1H) 3.64-3.76 (m, 1H)4.56 (d, J=8.49 Hz, 1H) 6.47 (s, 1H) 8.07 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamate

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.90-1.00 (m, 6H) 1.18-1.25 (m, 6H)1.34-1.36 (m, 9H) 1.69-1.90 (m, 1H) 3.34-3.82 (m, 6H) 4.53-4.77 (m, 1H)5.45-5.55 (m, 1H) 6.37 (dd, J=15.37, 6.59 Hz, 1H) 6.56 (s, 1H) 8.05 (s,1H). LCMS (ESI) 441 (M+H).

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.90 (d, J=6.73 Hz, 3H) 0.96(d, J=7.03 Hz, 3H) 1.55-1.66 (m, 10H) 4.14 (dd, J=13.61, 3.95 Hz, 1H)4.52-4.63 (m, 1H) 4.84 (dd, J=13.61, 1.32 Hz, 1H) 7.37 (s, 1H) 8.95 (s,1H). LCMS (ESI) 383 (M+H).

Compound 55

Compound 55 was synthesized using an analogous synthetic sequence asthat described for Compound 44. LCMS (ESI) 265 (M+H).

Example 56 Synthesis of Compound 56

Compound 56 was synthesized using 5-bromo-2,4-dichloro-pyrimidine andCompound 17 as starting materials, and following a similar sequence ofsynthetic steps as for Compound 55. The analytical data was consistentwith that described for its antipode (Compound 55). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 6H) 1.73-1.86 (m, 1H) 3.67-3.76 (m,2H) 4.11-4.21 (m, 1H) 7.13-7.19 (m, 1H) 8.56 (s, 1H) 9.05 (s, 1H). LCMS(ESI) 265 (M+H).

Example 57 Synthesis of Compound 57

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and tert-butylN-(2-amino-2-methyl-propyl)carbamate using analogous reaction conditionsas described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.LCMS (ESI) 379 (M+H).

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11-1.22 (m, 6H) 1.31-1.45 (m, 15H)3.10-3.24 (m, 2H) 3.51-3.76 (m, 4H) 5.60 (s, 1H) 6.94 (s, 1H) 7.33 (t,J=6.44 Hz, 1H) 8.18 (s, 1H). LCMS (ESI) 427 (M+H).

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.43 (s, 9H) 1.73 (s, 6H) 4.06 (s,2H) 7.46 (s, 1H) 9.23 (s, 1H). LCMS (ESI) 369 (M+H).

Compound 57

Compound 57 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.73 (s,6H) 3.50 (d, J=2.93 Hz, 2H) 7.25 (s, 1H) 8.46-8.55 (m, 1H) 9.07 (s, 1H).LCMS (ESI) 251 (M+H).

Example 58 Synthesis of Compound 58

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidine andIntermediate K using the analogous reaction conditions as described fortert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.18-1.54 (m, 17H) 2.23 (d,J=14.35 Hz, 2H) 3.36 (d, J=6.44 Hz, 2H) 5.82 (s, 1H) 6.93 (s, 1H) 8.22(s, 1H). LCMS (ESI) 419 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl]carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl]carbamate was synthesized using similar experimental conditions to thoseused in the synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.08-1.16 (m, 6H) 1.17-1.54 (m, 17H) 2.13(br. s., 2H) 3.36 (d, J=6.73 Hz, 2H) 3.50-3.69 (m, 4H) 5.72 (s, 1H) 6.94(s, 1H) 5.72 (br. s., 1H) 8.17 (s, 1H). LCMS (ESI) 467 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrol[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrol[2,3-d]pyrimidine-6-carboxylicacid was synthesized using analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.37-1.54 (m, 13H) 1.75 (br. s.,4H) 2.74 (br. s., 2H) 3.78-3.84 (m, 2H) 7.44-7.51 (m, 1H) 8.23 (s, 1H)9.11 (s, 1H). LCMS (ESI) 409 (M+H).

Compound 58

Compound 58 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.28 (br.s., 2H) 1.42 (br. s., 2H) 1.70 (br. s., 4H) 1.85-1.95 (m, 2H) 2.69 (m,2H) 7.16-7.25 (m, 1H) 8.41 (br. s., 1H) 9.04 (s, 1H). LCMS 291 (M+H).

Example 59 Synthesis of Compound 59

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate

tert-butyl N-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidineand Intermediate L using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.34 (s, 9H) 1.50-1.58 (m, 2H) 1.63-1.78(m, 4H) 1.96-2.06 (m, 2H) 3.25 (d, J=6.15 Hz, 2H) 6.71 (s, 1H) 7.18 (t,J=6.29 Hz, 1H) 8.20 (s, 1H). LCMS (ESI) 405 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 453 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.47 (s, 9H) 1.74 (br. s., 2H) 1.88(br. s., 2H) 2.04 (br. s., 2H) 2.41-2.45 (m, 2H) 4.06 (s, 2H) 7.45 (s,1H) 9.11 (s, 1H). LCMS (ESI) 395 (M+H).

Compound 59

Compound 59 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.72 (br.s., 2H) 1.86-1.93 (m, 2H) 1.99 (d, J=3.81 Hz, 2H) 2.40 (br. s., 2H) 3.48(d, J=2.34 Hz, 2H) 7.22 (s, 1H) 8.53 (br. s., 1H) 9.05 (s, 1H). LCMS(ESI) 277 (M+H).

Example 60 Synthesis of Compound 60

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl] carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Busing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.The analytical data is consistent with that described for theL-enantiomer.

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamate

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.21-1.31 (m, 12H) 1.38-1.46 (m,11H) 1.70 (m, 1H) 3.24 (m, 2H) 3.65-3.82 (m, 4H) 4.86 (br s., 1H), 5.65(s, 1H) 5.85 (br s., 1H) 6.94 (s, 1H) 8.21 (s, 1H). LCMS (ESI) 455(M+H).

7-[1-[(tert-butxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. The analytical data was consistent with that described for theL-isomer.

Compound 60

Compound 60 was synthesized using an analogous synthetic sequence asthat described for Compound 44. The analytical data was consistent withthat described for the L-isomer.

Example 61 Synthesis of Compound 61

To a solution of Compound 60 (100 mg, 0.00024 mole) in DMF (3.0 mL) wasadded sodium hydride (60% dispersion in oil), (27.6 mg, 3 eq). Afterstirring for 15 mins, methyl iodide (30, 2 eq) was added. The contentswere stirred at room temperature for 30 mins. After the addition of satNaHCO₃, ethyl acetate was added. Separation of the organic layerfollowed by drying with magnesium sulfate and concentration under vacuumafforded the product. Analytical data was similar to the Compound 49.

Example 62 Synthesis of Compound 62

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[(1S,2S)-2-aminocyclopentyl]carbamate with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.27 (s, 9H) 1.42-1.54 (m, 2H) 1.56-1.65(m, 2H) 1.80-1.88 (m, 1H) 1.96-2.01 (m, 1H) 3.88-3.96 (m, 1H) 4.03-4.09(m, 1H) 6.91 (d, J=8.20 Hz, 1H) 7.41 (d, J=7.32 Hz, 1H) 8.18 (s, 1H).LCMS (ESI) 391 (M+H).

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.13 (t, 6H) 1.28 (s, 9H) 1.42-1.52 (m,2H) 1.58-1.65 (m, 2H) 1.81-1.90 (m, 1H) 1.99-2.08 (m, 1H) 3.49-3.60 (m,2H) 3.63-3.71 (m, 2H) 3.84-3.93 (m, 1H) 3.96-4.04 (m, 1H) 5.53 (s, 1H)6.96 (d, J=7.90 Hz, 1H) 7.34 (d, J=7.03 Hz, 1H) 8.14 (s, 1H). LCMS (ESI)439 (M+H).

7-[(1S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.41-1.52 (m, 9H) 1.55-1.68 (m, 1H)1.88-2.00 (m, 2H) 2.05-2.15 (m, 1H) 2.26-2.35 (m, 1H) 2.71-2.89 (m, 1H)4.01-4.16 (m, 1H) 4.28-4.45 (m, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(ESI) 381 (M+H).

Compound 62

Compound 62 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.48-1.60(m, 1H) 1.88-1.98 (m, 3H) 1.99-2.08 (m, 1H) 2.66-2.75 (m, 1H) 3.63-3.74(m, 1H) 3.99-4.12 (m, 1H) 7.21 (s, 1H) 8.89 (s, 1H) 9.04 (s, 1H). LCMS(ESI) 263 (M+H).

Example 63 Synthesis of Compound 63

To chloro tricycliclactam (0.050 g, 0.225 mmole) in dioxane (2.0 mL)under nitrogen was added 5-(4-methylpiperazin-1-yl)pyridin-2-amine(0.052 g, 1.2 eq, 0.270 mmole) followed by the addition of Pd₂(dba)₃(18.5 mg), BINAP (25 mg) and sodium-tert-butoxide (31 mg, 0.324 mmole).The contents of the flask are degassed for 10 minutes and then heated to100 degrees for 12 hours. The crude reaction was loaded on a silica gelcolumn and eluted with DCM/MeOH (0-15%) to afford the desired product(26 mg). To this compound dissolved in DCM/MeOH (10%) was added 3N HClin iso-propanol (2 eq) and the reaction was stirred overnight.Concentration under vacuum afforded the hydrochloride salt. ¹HNMR(d6-DMSO) δ ppm 11.13 (brs, 1H), 9.07 (s, 1H), 8.42 (s, 1H), 8.03 (br m1H), 7.99 (s, 1H), 7.67 (brm, 1H), 7.18 (s, 1H), 4.33 (m, 2H), 3.79 (m,2H), 3.64 (m, 2H), 3.50 (m, 2H), 3.16 (m, 4H), 2.79 (s, 3H). LCMS (ESI)379 (M+H).

Example 64 Synthesis of Compound 64

To chloro tricycliclactam (0.075 g, 0.338 mmole) in dioxane (3.5 mL)under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate (0.098 g, 1.05 eq)followed by the addition of Pd₂(dba)₃ (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude reaction was loaded onto a silica gel column and elutedwith DCM/MeOH (0-10%) to afford the desired product (32 mg). ¹HNMR(d6-DMSO) δ ppm 9.48 (s, 1H), 8.84 (s, 1H), 8.29 (s, 1H), 8.18 (s, 1H),7.99 (s, 1H), 7.42 (m, 1H), 6.98 (s, 1H), 4.23 (m, 2H), 3.59 (m, 2H),3.45 (m, 4H), 3.50 (m, 2H), 3.05 (m, 4H). LCMS (ESI) 465 (M+H).

Example 65 Synthesis of Compound 65

To a solution of Compound 64 (23 mg) in 10% DCM/MeOH was added 10 mL ofa 3M solution of HCl in iso-propanol. The contents were stirred for 16hrs. Concentration of the reaction mixture afforded the hydrochloridesalt. ¹HNMR (d6-DMSO) δ ppm 9.01 (s, 1H), 7.94 (m, 1H), 7.86 (m, 1H),7.23 (s, 1H), 4.30 (m, 2H), 3.64 (m, 2H), 3.36 (m, 4H), 3.25 (m, 4H).LCMS (ESI) 465 (M+H).

Example 66 Synthesis of Compound 66

To chloro-N-methyltricyclic amide (0.080 g, 0.338 mmole) in dioxane (3.5mL) under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate 0.102 g (1.1 eq) followedby the addition of Pd₂(dba)₃ (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude product was purified using silica gel columnchromatography with an eluent of dichloromethane/methanol (0-5%) toafford the desired product (44 mg). ¹HNMR (d6-DMSO) δ ppm 9.49 (s, 1H),8.85 (s, 1H), 8.32 (m, 1H), 8.02 (s, 1H), 7.44 (m, 1H), 7.00 (s, 1H),4.33 (m, 2H), 3.80 (m, 2H), 3.48 (m, 4H), 3.07 (m, 4H), 3.05 (s, 3H),1.42 (s, 9H). LCMS (ESI) 479 (M+H).

Example 67 Synthesis of Compound 67

To Compound 66 (32 mg) was added 3N HCL (10 mL) in isopropanol and thecontents were stirred at room temperature overnight for 16 hrs.Concentration afforded the hydrochloride salt. ¹HNMR (d6-DMSO) δ ppm9.13 (m, 2H), 8.11 (m, 1H), 8.10 (s, 1H), 7.62 (m, 1H), 7.21 (s, 1H),4.43 (m, 2H), 3.85 (m, 2H), 3.41 (m, 4H), 3.28 (m, 4H), 3.08 (s, 3H).LCMS (ESI) 379 (M+H).

Example 68 Synthesis of Compound 68

Compound 68 was synthesized using similar experimental conditions tothat described for compound 64. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d,J=7.03 Hz, 3H) 1.01 (d, J=6.73 Hz, 3H) 1.35-1.48 (m, 9H) 2.16 (dd,J=14.64, 6.73 Hz, 1H) 3.00-3.14 (m, 4H) 3.40-3.51 (m, 4H) 3.51-3.60 (m,1H) 3.63-3.74 (m, 1H) 4.44 (dd, J=7.90, 3.81 Hz, 1H) 6.99 (s, 1H) 7.46(dd, J=8.93, 2.78 Hz, 1H) 7.94-8.09 (m, 2H) 8.31 (dd, J=9.08, 1.46 Hz,1H) 8.85 (s, 1H) 9.46 (s, 1H). LCMS (ESI) 507 (M+H).

Example 69 Synthesis of Compound 69

Compound 69 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77-0.86 (m, 3H) 0.96 (d, J=7.03 Hz, 3H)2.10-2.24 (m, 1H) 3.07 (s, 3H) 3.37-3.79 (m, 8H) 4.00 (dd, J=13.61, 4.54Hz, 2H) 4.63-4.73 (m, 1H) 7.20 (s, 1H) 7.58-7.71 (m, 1H) 7.99 (d, J=2.34Hz, 1H) 8.12 (d, J=9.37 Hz, 1H) 9.11 (s, 1H) 9.41 (br. s., 2H) 11.76(br. s., 1H). LCMS (ESI) 421 (M+H).

Example 70 Synthesis of Compound 70

Compound 70 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. The characterization data (NMR and LCMS) was consistent with thatreported for compound 71.

Example 71 Synthesis of Compound 71

Compound 71 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d, J=6.73 Hz, 3H) 1.01 (d,J=6.73 Hz, 3H) 2.18 (dd, J=14.49, 7.17 Hz, 1H) 3.18-3.84 (m, 10H)4.53-4.71 (m, 1H) 7.24 (s, 1H) 7.65 (d, J=9.37 Hz, 1H) 8.01 (d, J=2.64Hz, 1H) 8.14 (d, J=1.46 Hz, 1H) 8.35 (d, J=5.27 Hz, 1H) 9.14 (s, 1H)9.46 (s, 2H) 11.80 (s, 1H) LCMS (ESI) 407 (M+H).

Example 72 Synthesis of Compound 72 (Compound UUU)

Compound 72 was synthesized using similar experimental conditions tothat described for compounds 64 and 65 and was recovered as an HCl salt.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77 (d, J=7.03 Hz, 3H) 0.99 (d, J=6.73Hz, 3H) 2.10-2.24 (m, 1H) 3.18-3.81 (m, 10H) 4.54-4.69 (m, 1H) 7.22 (s,1H) 7.63 (d, J=9.08 Hz, 1H) 7.99 (d, J=2.63 Hz, 1H) 8.11 (s, 1H) 8.33(d, J=5.27 Hz, 1H) 9.12 (s, 1H) 9.43 (s, 2H) 11.77 (s, 1H). LCMS (ESI)407 (M+H).

Example 73 Synthesis of Compound 73

Compound 73 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.84 (d, J=6.73 Hz, 3H) 0.98 (d,J=6.73 Hz, 3H) 2.12-2.26 (m, 1H) 3.09 (s, 3H) 3.22-3.81 (m, 8H) 4.01(dd, J=13.61, 4.25 Hz, 2H) 4.59-4.72 (m, 1H) 7.19 (s, 1H) 7.74 (s, 1H)7.96-8.10 (m, 2H) 9.08 (s, 1H) 9.22 (s, 2H). LCMS (ESI) 421 (M+H).

Example 74 Synthesis of Compound 74

Compound 74 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=4.98 Hz, 3H) 0.95 (d, J=4.98 Hz, 3H)1.42-1.70 (m, 3H) 2.77 (d, J=2.93 Hz, 3H) 3.07-4.14 (m, 10H) 4.95 (s,1H) 7.20 (s, 1H) 7.66 (d, J=9.66 Hz, 1H) 7.94 (s, 1H) 8.08-8.16 (m, 1H)8.33 (d, J=4.68 Hz, 1H) 9.09 (s, 1H) 11.38 (s, 1H) 11.71 (s, 1H). LCMS(ESI) 435 (M+H).

Example 75 Synthesis of Compound 75

Compound 75 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.87 (d, J=6.15 Hz, 3H) 0.94 (d,J=6.15 Hz, 3H) 1.57 (d, J=84.61 Hz, 3H) 3.05 (s, 3H) 3.13-3.55 (m, 8H)3.69 (d, J=78.17 Hz, 2H) 4.90 (s, 1H) 7.15 (s, 1H) 7.63-7.85 (m, 1H)7.93 (s, 1H) 8.26 (s, 1H) 9.03 (s, 1H) 9.20 (s, 2H). LCMS (ESI) 421(M+H).

Example 76 Synthesis of Compound 76

Compound 76 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=6.44 Hz, 3H) 0.95 (d, J=6.44 Hz, 3H)1.43-1.70 (m, 3H) 2.78 (d, J=2.93 Hz, 3H) 3.05 (s, 3H) 3.24-3.84 (m, 8H)4.01 (d, J=9.66 Hz, 2H) 4.89-5.01 (m, 1H) 7.15 (s, 1H) 7.77 (s, 1H)7.91-8.05 (m, 2H) 9.03 (s, 1H) 10.96-11.55 (m, 2H). LCMS (ESI) 449(M+H).

Example 77 Synthesis of Compound 77

Compound 77 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.83-0.88 (d, J=6.15 Hz, 3H) 0.95(d, J=6.15 Hz, 3H) 1.40-1.71 (m, 3H) 3.28-3.83 (m, 8H) 4.00 (d, J=3.22Hz, 2H) 4.91-5.08 (m, 1H) 7.17 (s, 1H) 7.68 (d, J=9.66 Hz, 1H) 7.93 (s,1H) 8.07 (s, 1H) 9.06 (s, 1H) 9.40 (s, 2H) 11.59 (s, 1H). LCMS (ESI) 435(M+H).

Example 78 Synthesis of Compound 78

To Compound 50 0.060 g (0.205 mmole) was added5-(4-methylpiperazin-1-yl)pyridin-2-amine (35.42 mg, 0.9 eq) followed bythe addition of 1,4-dioxane (3 mL). After degassing with nitrogen,Pd₂dba₃ (12 mg), BINAP (16 mg) and sodium tert-butoxide (24 mg) wereadded. The contents were then heated at 90 degrees in a CEM Discoverymicrowave for 3 hrs. The reaction was then loaded onto a silica gelcolumn and purified by eluting with DCM/MeOH (0-15%). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.75 (t, J=7.47 Hz, 3H) 0.91 (d, J=6.73 Hz, 3H) 1.04-1.20(m, 2H) 1.80-1.98 (m, 1H) 2.77 (d, J=3.81 Hz, 3H) 2.94-3.90 (m, 10H)4.54-4.68 (m, 1H) 7.06-7.23 (m, 2H) 7.56-7.75 (m, 1H) 7.90-8.12 (m, 2H)8.29 (s, 1H) 9.07 (s, 1H) 10.98-11.74 (m, 2H). LCMS (ESI) 435 (M+H).

Example 79 Synthesis of Compound 79

Compound 79 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.75(t, J=7.32 Hz, 3H) 0.90 (d, J=6.73 Hz, 3H) 1.07-1.15 (m, 2H) 1.85-1.94(m, 1H) 3.17-3.75 (m, 10H) 4.58-4.67 (m, 1H) 7.17 (s, 1H) 7.71 (s, 1H)7.96 (s, 1H) 7.98-8.05 (m, 1H) 8.28 (d, J=4.10 Hz, 1H) 9.06 (s, 1H) 9.39(s, 2H). LCMS (ESI) 421 (M+H).

Example 80 Synthesis of Compound 80

Compound 80 was synthesized in a similar manner to that described forcompound 78. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78 (t, J=7.32 Hz, 3H) 0.86(d, J=6.73 Hz, 3H) 1.13-1.21 (m, 2H) 1.84-1.96 (m, 1H) 2.77 (d, J=4.39Hz, 3H) 3.04 (s, 3H) 3.11-3.84 (m, 8H) 3.98 (dd, J=13.61, 4.25 Hz, 2H)4.66-4.74 (m, 1H) 7.17 (s, 1H) 7.64 (s, 1H) 7.96 (d, J=2.34 Hz, 1H)8.03-8.13 (m, 1H) 9.08 (s, 1H) 11.26 (s, 1H) 11.66 (s, 1H). LCMS (ESI)449 (M+H).

Example 81 Synthesis of Compound 81

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78(t, J=7.32 Hz, 3H) 0.85 (d, J=6.73 Hz, 3H) 1.10-1.27 (m, 2H) 1.82-1.99(m, 1H) 3.04 (s, 3H) 3.28-3.77 (m, 8H) 3.97 (dd, J=13.91, 4.54 Hz, 2H)4.62-4.75 (m, 1H) 7.07-7.24 (m, 1H) 7.62-7.75 (m, 1H) 7.94 (d, J=2.34Hz, 1H) 7.97-8.08 (m, 1H) 9.05 (s, 1H) 9.29 (s, 2H). LCMS (ESI) 435(M+H).

Example 82 Synthesis of Compound 82

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.96(s, 9H) 3.15-3.87 (m, 10H) 4.42-4.53 (m, 1H) 6.99 (s, 1H) 7.24 (s, 1H)8.06 (s, 1H) 8.11-8.21 (m, 1H) 8.79-8.98 (m, 2H) 9.25 (s, 2H) 9.88 (s,1H). LCMS (ESI) 421 (M+H).

Example 83 Synthesis of Compound 83

Compound 83 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.95(s, 9H) 2.79 (d, J=4.10 Hz, 3H) 3.06-3.86 (m, 10H) 4.56-4.67 (m, 1H)7.17 (s, 1H) 7.70 (s, 1H) 7.96 (d, J=2.63 Hz, 1H) 7.99-8.08 (m, 1H) 8.26(s, 1H) 9.06 (s, 1H) 10.80 (s, 1H). LCMS (ESI) 435 (M+H).

Example 84 Synthesis of Compound 84

Compound 84 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 2.75-2.81 (m, 3H) 3.12-3.16 (m, 2H) 3.46-3.54 (m, 4H) 3.60-3.69 (m,2H) 3.72-3.79 (m, 1H) 4.07-4.18 (m, 2H) 6.06-6.09 (m, 1H) 6.90 (d,J=7.61 Hz, 2H) 7.20-7.31 (m, 3H) 7.33 (s, 1H) 7.49-7.55 (m, 1H)7.62-7.70 (m, 1H) 7.92 (d, J=2.93 Hz, 1H) 8.22 (s, 1H) 9.14 (s, 1H).LCMS (ESI) 455 (M+H).

Example 85 Synthesis of Compound 85

Compound 85 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.21(s, 4H) 3.35-3.67 (m, 5H) 4.07-4.20 (m, 2H) 6.13 (s, 1H) 6.90 (d, J=7.32Hz, 2H) 7.22-7.31 (m, 3H) 7.36 (s, 1H) 7.48 (d, J=9.37 Hz, 1H) 7.93 (d,J=2.34 Hz, 1H) 8.04-8.11 (m, 1H) 8.25 (d, J=4.98 Hz, 1H) 9.17 (s, 1H)11.77 (br, s., 1H). LCMS (ESI) 441 (M+H).

Example 86 Synthesis of Compound 86

Compound 86 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.90(d, J=6.15 Hz, 6H) 1.72-1.89 (m, 1H) 3.15-3.92 (m, 9H) 4.10-4.46 (m, 2H)7.18 (s, 1H) 7.59 (d, J=8.78 Hz, 1H) 8.00 (s, 1H) 8.13 (d, J=9.37 Hz,1H) 8.55 (s, 1H) 9.09 (s, 1H) 9.67 (s, 2H) 11.91 (s, 1H). LCMS (ESI) 407(ESI).

Example 87 Synthesis of Compound 87

Compound 87 was synthesized in a manner similar to compound 86 and wasconverted to an HCl salt. The characterization data (NMR and LCMS) wassimilar to that obtained for the antipode compound 86.

Example 88 Synthesis of Compound 88

Compound 88 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.78(s, 6H) 3.40-3.53 (m, 6H) 3.64-3.73 (m, 4H) 7.27 (s, 1H) 7.66 (d, J=9.37Hz, 1H) 7.98 (d, J=2.34 Hz, 1H) 8.12 (br. s., 1H) 8.47 (br. s., 1H) 9.11(s, 1H) 9.45 (br. s., 2H) 11.62 (br. s., 1H). LCMS (ESI) 393 (M+H).

Example 89 Synthesis of Compound 89 (Also Referred to as Compound T)

Compound 89 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.47 (br. s., 6H) 1.72 (br. s., 2H) 1.92 (br. s., 2H) 2.77 (br. s.,3H) 3.18 (br. s., 2H) 3.46 (br. s., 2H) 3.63 (br. s., 2H) 3.66 (d,J=6.15 Hz, 2H) 3.80 (br. s., 2H) 7.25 (s, 1H) 7.63 (br. s., 2H) 7.94(br. s., 1H) 8.10 (br. s., 1H) 8.39 (br. s., 1H) 9.08 (br. s., 1H) 11.59(br. s., 1H). LCMS (ESI) 447 (M+H).

Example 90 Synthesis of Compound 90 (Also Referred to as Compound Q)

Compound 90 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.27-1.64 (m, 6H) 1.71 (br. s., 2H) 1.91 (br. s., 2H) 2.80 (br. s., 1H)3.17-3.24 (m, 2H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 7.26 (br. s., 1H)7.63 (br. s., 1 H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40 (br. s., 1H)9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H). LCMS (ESI) 433(M+H).

Example 91 Synthesis of Compound 91 (Also Referred to as Compound ZZ)

Compound 91 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.75 (m, 2H) 1.83-1.92 (m, 2H) 1.96-2.06 (m, 2H)2.49-2.58 (m, 2H) 2.79 (d, J=3.81 Hz, 3H) 3.06-3.18 (m, 4H) 3.59-3.69(m, 2H) 3.73-3.83 (m, 2H) 4.04-4.12 (m, 2H) 7.17 (br. s., 1H) 7.60-7.70(m, 2H) 7.70-7.92 (m, 2H) 7.96 (br. s., 1H) 8.41 (br. s., 1H) 8.98 (br.s., 1H) 10.77 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 92 Synthesis of Compound 92

Compound 92 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.64-1.75 (m, 2H) 1.84-1.92 (m, 2H) 1.96-2.05 (m, 2H) 2.48-2.56 (m, 2H)3.22 (br. s., 4H) 3.42-3.48 (m, 4H) 3.60-3.69 (m, 2H) 4.05-4.13 (m, 1H)7.18 (s, 1H) 7.65 (d, J=13.47 Hz, 1H) 7.70-7.77 (m, 1H) 7.94 (d, J=1.76Hz, 1H) 8.42 (br. s., 1H) 9.00 (s, 1H) 9.15 (br. s., 2H). LCMS (ESI) 419(M+H).

Example 93 Synthesis of Compound 93

Compound 93 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.76(br. s., 2H) 1.89 (br. s., 2H) 2.03 (br. s., 2H) 2.47-2.58 (m, 2H) 3.04(s, 3H) 3.22 (br. s., 4H) 3.39 (br. s., 4H) 3.66 (s, 2H) 7.21 (s, 1H)7.67 (d, J=9.37 Hz, 1H) 7.93 (br. s., 1H) 7.98-8.09 (m, 1H) 9.04 (s, 1H)9.34 (br. s., 2H) 11.31 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 94 Synthesis of Compound 94

Compound 94 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.66-1.77 (m, 2H) 1.84-1.94 (m, 2H) 1.96-2.08 (m, 2H)2.48-2.57 (m, 2H) 3.36-3.52 (m, 4H) 3.60-3.80 (m, 6H) 7.21 (s, 1H)7.53-7.74 (m, 2H) 7.86 (s, 1H) 8.02 (s, 1H) 8.45 (s, 1H) 9.03 (s, 1H)11.19 (br. s., 1H). LCMS (ESI) 420 (M+H).

Example 95 Synthesis of Compound 95

Compound 95 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.79 (m, 2H) 1.85-1.95 (m, 2H) 1.97-2.08 (m, 2H)2.47-2.54 (m, 2H) 3.40-3.58 (m, 5H) 3.65 (dd, J=21.67, 5.56 Hz, 1H)3.69-3.78 (m, 4H) 7.24 (s, 1H) 7.97-8.17 (m, 2H) 8.48 (s, 1H) 9.08 (s,1H) 11.81 (s, 1H). LCMS (ESI) 421 (M+H).

Example 96 Synthesis of Compound 96

Compound 96 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.55-1.74 (m, 2H) 1.80-1.98 (m, 4H) 2.48-2.60 (m, 2H)3.40-3.50 (m, 4H) 3.57-3.72 (m, 2H) 3.90-4.20 (m, 4H) 7.08 (s, 1H)7.37-7.57 (m, 2H) 7.70 (m, 2H) 8.32 (s, 1H) 8.88 (s, 1H) 9.98 (s, 1H).LCMS (ESI) 419 (M+H).

Example 97 Synthesis of Compound 97 (Also Referred to as Compound III)

Compound 97 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30 (d, J=5.27 Hz, 6H) 1.65-1.78 (m, 2H) 1.83-1.95 (m,2H) 1.97-2.10 (m, 2H) 2.45-2.55 (m, 2H) 3.25-3.36 (m, 1H) 3.39-3.48 (m,4H) 3.60-3.70 (m, 4H) 3.75-4.15 (m, 2H) 7.24 (s, 1H) 7.54-7.75 (m, 2H)7.95 (s, 1H) 8.10 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 11.25 (s, 1H) 11.48(s, 1H). LCMS (ESI) 461 (M+H).

Example 98 Synthesis of Compound 98

Compound 98 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.99 (d, J=6.15 Hz, 6H) 1.65-1.78 (m, 2H) 1.90 (m, 2H)1.97-2.08 (m, 2H) 2.08-2.17 (m, 1H) 2.45-2.55 (m, 2H) 2.88-3.02 (m, 2H)3.33-3.48 (m, 4H) 3.50-3.90 (m, 6H) 7.24 (s, 1H) 7.67 (s, 2H) 7.94 (s,1H) 8.12 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 10.77 (s, 1H) 11.51 (s, 1H).LCMS (ESI) 475 (M+H).

Example 99 Synthesis of Compound 99

Compound 99 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.13 (d, J=5.86 Hz, 6H) 1.66-1.77 (m, 2H) 1.84-1.94 (m,2H) 1.97-2.09 (m, 2H) 2.40-2.53 (m, 2H) 3.37-3.49 (m, 2H) 3.50-3.59 (m,2H) 3.59-3.73 (m, 4H) 7.23 (s, 1H) 7.64 (m, 3H) 7.85 (s, 1H) 8.11 (s,1H) 8.47 (s, 1H) 9.05 (s, 1H). 11.35 (br s., 1H). LCMS (ESI) 448 (M+H).

Example 100 Synthesis of Compound 100

Compound 100 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.50-1.57 (m, 2H) 1.62-1.68 (m, 3H) 1.68-1.75 (m, 2H)1.84-1.92 (m, 2H) 1.97-2.08 (m, 2H) 2.48-2.53 (m, 2H) 3.14-3.23 (m, 4H)3.43-3.47 (m, 2H) 3.58-3.70 (m, 2H) 7.22 (s, 1H) 7.58-7.70 (m, 2H)7.85-8.00 (m, 1H) 8.16 (d, 1H) 8.46 (s, 1H) 9.04 (s, 1H) 11.37 (br s.,1H). LCMS (ESI) 418 (M+H).

Example 101 Synthesis of Compound 101 (Also Referred to as Compound WW)

Compound 101 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. 1HNMR (600 MHz,DMSO-d₆) δ ppm 1.72 (s, 2H) 1.90 (s, 4H) 2.03 (s, 2H) 2.21 (s, 2H)2.48-2.54 (m, 2H) 2.73 (s, 2H) 3.03 (s, 2H) 3.25-3.35 (m, 1H) 3.38-3.48(m, 4H) 3.65-3.99 (m, 5H) 7.23 (s, 1H) 7.63 (d, J=9.66 Hz, 1H) 7.90 (s,1H) 8.13 (s, 1H) 8.47 (s, 1H) 9.06 (s, 1H) 10.50 (br s., 1H). LCMS (ESI)503 (M+H).

Example 102 Synthesis of Compound 102 (Also Referred to as Compound HHH)

Compound 102 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. 1HNMR (600 MHz,DMSO-d₆) δ ppm 1.63-1.85 (m, 6H) 1.87-1.92 (m, 2H) 1.99-2.06 (m, 2H)2.15-2.23 (m, 2H) 2.47-2.53 (m, 1H) 2.69-2.79 (m, 2H) 2.81-2.91 (m, 2H)2.98-3.08 (m, 2H) 3.32-3.48 (m, 4H) 3.57-3.72 (m, 4H) 3.77-3.85 (m, 2H)7.22 (s, 1H) 7.60-7.68 (m, 2H) 7.90 (s, 1H) 8.07 (s, 1H) 8.46 (s, 1H)9.04 (s, 1H). 11.41 (br s., 1H). LCMS (ESI) 501 (M+H).

Example 103 Synthesis of Compound 103

Compound 103 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.76 (m, 2H) 1.87-1.93 (m, 2H) 2.00-2.07 (m, 2H)2.48-2.53 (m, 2H) 2.67-2.72 (m, 4H) 3.44-3.47 (m, 2H) 3.50-3.55 (m, 4H)7.24 (s, 1H) 7.61 (d, J=9.37 Hz, 2H) 7.86 (d, J=2.63 Hz, 1H) 8.09 (d,J=12.88 Hz, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.41 (br s., 1H). LCMS (ESI)436 (M+H).

Example 104 Synthesis of Compound 104

Compound 104 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.29 (d, J=6.73 Hz, 6H) 1.66-1.79 (m, 2H) 1.84-1.95 (m,2H) 1.98-2.09 (m, 2H) 2.46-2.55 (m, 2H) 3.29-3.39 (m, 2H) 3.58-3.70 (m,4H) 3.77-3.86 (m, 4H) 7.24 (s, 1H) 7.66 (d, J=9.37 Hz, 1H) 7.96 (d,J=2.93 Hz, 1H) 8.08 (s, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 9.28 (s, 1H) 9.67(s, 1H) 11.36 (s, 1H). LCMS (ESI) 447 (M+H).

Example 105 Synthesis of Compound 105

Compound 105 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.73 (s, 2H) 1.76-1.85 (m, 2H) 1.85-1.94 (m, 2H)1.98-2.07 (m, 2H) 2.19-2.26 (m, 2H) 2.48-2.52 (m, 1H) 2.70-2.81 (m, 4H)3.13-3.20 (m, 1H) 3.30-3.48 (m, 3H) 3.58-3.71 (m, 4H) 3.78-3.84 (m, 4H)7.24 (s, 1H) 7.62 (d, J=9.37 Hz, 2H) 7.89 (d, J=1.17 Hz, 1H) 8.09-8.18(m, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.46 (br s., 1H). LCMS (ESI) 519(M+H).

Example 106 Synthesis of Compound 106

Compound 106 was synthesized using similar conditions to those describedfor compound 78 followed by the deblocking step described for compound65 and was converted to an HCl salt. 1HNMR (600 MHz, DMSO-d₆) δ ppm1.65-1.75 (m, 2H) 1.85-1.93 (m, 2H) 1.93-1.99 (m, 1H) 2.00-2.06 (m, 2H)2.08-2.14 (m, 1H) 2.47-2.55 (m, 2H) 3.07-3.25 (m, 2H) 3.25-3.69 (m, 5H)4.46 (s, 1H) 4.67 (s, 1H) 7.22 (s, 1H) 7.58-7.69 (m, 2H) 8.46 (s, 1H)9.02 (s, 1H) 9.34 (s, 1H) 9.65 (s, 1H). LCMS (ESI) 431 (M+H).

Example 107 Synthesis of Compound 107 (Also Referred to as Compound YY)

Compound 107 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. 1HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.82 (m, 3H) 1.89 (br. s., 2H) 1.98-2.08 (m, 2H)2.13 (br. s., 2H) 2.47-2.55 (m, 2H) 2.68 (d, J=4.98 Hz, 6H) 2.71-2.80(m, 2H) 3.29-3.71 (m, 10H) 7.16-7.26 (m, 1H) 7.67 (d, J=9.66 Hz, 2H)7.91 (d, J=2.05 Hz, 1H) 8.14 (br. s., 1H) 8.48 (br. s., 1H) 9.05 (s, 1H)11.14 (br. s., 1H) 11.43 (br. s., 1H). LCMS (ESI) 461 (M+H).

Example 108 Synthesis of Compound 108

Compound 108 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 109 Synthesis of Compound 109

Compound 109 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 110 Synthesis of Compound 110

Compound 110 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.50-1.65 (m, 1H) 1.92-2.02 (m, 3H) 2.06-2.15 (m,1H) 2.78 (d, J=3.81 Hz, 4H) 3.10-3.20 (m, 4H) 3.47-3.51 (m, 2H)3.64-3.71 (m, 1H) 3.76-3.83 (m, 2H) 3.98-4.14 (m, 1H) 7.20 (s, 2H) 7.77(s, 1H) 7.97 (s, 2H) 8.81 (s, 1H) 9.03 (s, 1H) 10.97 (br s., 1H). LCMS(ESI) 419 (M+H).

Example 111 Synthesis of Compound 111

Compound 111 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.54-1.59 (m, 1H) 1.92-2.01 (m, 3H) 2.06-2.15 (m,1H) 2.76-2.84 (m, 1H) 3.17-3.24 (m, 6H) 3.64-3.71 (m, 2H) 4.02-4.11 (m,2H) 7.22 (s, 2H) 7.64 (s, 1H) 7.97 (s, 2H) 8.75 (s, 1H) 8.97 (s, 1H)9.21 (s, 1H). LCMS (ESI) 405 (M+H).

Example 112 Synthesis of Compound 112

Compound 112 was synthesized using similar experimental conditions tothat described for compound 64.

Example 113 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate, Compound113

To a solution of 5-bromo-2,4-dichloropyrimidine (12.80 g, 0.054 mole) inethanol (250 mL) was added Hunig's base (12.0 mL) followed by theaddition of a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (10g, 0.0624 mole) in ethanol (80 mL). The contents were stirred overnightfor 20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (800mL) and water (300 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)351 (M+H).

Example 114 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate, Compound 114

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (5 g, 14.23mmole) in toluene (42 mL) and triethylamine (8.33 mL) under nitrogen wasadded triphenyl arsine (4.39 g), 3,3-diethoxyprop-1-yne (3.24 mL) andPddba (1.27 g). The contents were heated at 70 degrees for 24 hrs. Afterfiltration through CELITE®, the crude reeacton was columned usinghexane/ethyl acetate (0-20%) to afford the desired product 3.9 g).Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.LCMS (ESI) 399 (M+H).

Example 115 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 115

To a solution of Compound 114 (3.9 g, 0.00976 mole) in THF (60 mL) wasadded TBAF (68.3 mL, 7 eq). The contents were heated to 45 degrees for 2hrs. Concentration followed by column chromatography using ethylacetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d6-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 116 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 116

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.00025 mol) in acetonitrile (2 mL) was added1,3-diiodo-5,5-dimethylhydantoin (95 mg, 1 eq), and solid NaHCO₃ (63 mg,3 eq). The reacation was stirred at room tempreature for 16 hrs. Thereaction was filtered and concentrated in vacuo. The product waspurified by silica gel column chromatography using hexane/ethylacetate(0-50%) to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale yellow solid (0.03 g). LCMS (ESI) 525 (M+H).

Example 117 Synthesis of tert-ButylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 117

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.19 mmole) in dioxane (3 mL) was added 2-methylphenylboronicacid (28 mg), tetrakis(triphenylphosphine)palladium (25 mg) andpotassium phosphate (250 mg) in water (0.3 mL). The reaction was heatedin a CEM Discovery microwave at 90° C. for 3 hrs. The crude reaction wasloaded onto silica gel and columned using hexane/ethyl acetate (0-30%)to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.06 g). LCMS (ESI) 489 (M+H).

Example 118 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 118

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.85 g, 1.74 mmole) in AcOH (10 mL) was added water (1.5 mL). Thereation was stirred at room tempreature for 16 hrs. The crude reactionwas then concentrated under vacuum. After the addition of ethyl acetate(50 mL), the organic layer was washed with satd. NaHCO₃. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum to afford the crude intermediate, tert-butylN-[2-[2-chloro-6-formyl-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.To this crude intermediate in DMF (5 mL) was added oxone (1.3 g). Afterstirring for 2.5 hrs, water (20 mL) and ethyl acetate (100 mL) wereadded. The organic layer was separated, dried and then concentratedunder vacuum to afford the crude product which was columned over silicagel using hexane/ethyl acetate (0-50%) to afford7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.112 g). LCMS (ESI) 431 (M+H).

Example 119 Synthesis of Compound 119

To7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrol[2,3-d]pyrimidine-6-carboxylicacid (0.1 g, 0.261 mmol) in DCM (4.1 mL) was added DMAP (20 mg) followedby the addition of N,N′-diisopropylcarbodiimide (0.081 mL, 2 eq). Afterstirring for 3 hrs, TFA (0.723 mL) was added. Stirring was thencontinued for another 30 minutes. The reaction mixture was neutralizedwith satd. NaHCO₃. DCM (20 mL) was then added and the organic layerseparated, dried with magnesium sulfate and then concentrated undervacuum to afford the crude product which was columned usinghexane/ethylacetate (0-100%) to afford chloro tricyclic amide Compound119 (0.65 g). LCMS (ESI) 313 (M+H).

Example 120 Synthesis of Compound 120

To the chloro tricyclic amide (0.040 g, 0.128 mmole) (Compound 119) indioxane (2.5 mL) under nitrogen was added Pd₂(dba)₃ (12 mg), sodiumtert-butoxide (16 mg), BINAP (16 mg) and 4-morpholinoaniline (22.7 mg, 1eq). The reaction mixture was heated at 90° C. in a CEM Discoverymicrowave for 3.0 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-6%) to afford theproduct (10 mg). LCMS (ESI) 455 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm2.14 (s, 3H) 3.23-3.50 (m, 2H) 3.57-3.73 (m, 2H), 3.81-3.92 (m, 8H),7.11-7.31 (m, 4H) 7.31-7.48 (m, 1H) 7.58-7.73 (m, 1H) 7.77-7.95 (m, 2H)8.05-8.21 (m, 1H) 8.44 (s, 1H) 9.85-10.01 (m, 1H).

Example 121 Synthesis of Compound 121

To the chloro tricyclic amide (0.024 g) (Compound 119) inN-methyl-2-pyrrolidone (NMP) (1.5 mL) was addedtrans-4-aminocyclohexanol (0.0768 mmol, 26.54 mg, 3 eq) and Hunig's base(0.4 mL). The reaction was heated in a CEM Discovery microwave vessel at150° C. for 1.2 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-10%) to afford theproduct (21 mg). LCMS (ESI) 392 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.23 (d, J=8.78 Hz, 4H) 1.84 (br. s., 4H) 2.11 (s, 3H) 3.34-3.43 (m, 1H)3.55 (br. s., 2H) 3.72 (br. s., 1H) 4.13 (br. s., 2H) 4.50 (br. s., 1H)7.03 (br. s., 1H) 7.12-7.28 (m, 4H) 7.96 (br. s., 1H) 8.18 (br. s., 1H).

Example 122 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 122

7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 341 (M+H).

Example 123 Synthesis of Compound 123

Chloro tricyclic amide, Compound 123, was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide (Compound 119). LCMS (ESI) 223 (M+H).

Example 124 Synthesis of Compound 124

To the chloro tricyclic amide, Compound 123 (0.035 g, 0.00157 mole) inNMP (1.5 mL) was added Hunig's base (0.3 mL) followed by the addition ofthe trans-4-aminocyclohexanol (54.2 mg). The reaction mixture was heatedat 150° C. for 1.5 hrs. The crude reaction was loaded onto a silica gelcolumn and the column was eluted with DCM/MeOH (0-10%) to afford theproduct (5 mg). LCMS (ESI) 302 (M+H).

Example 125 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate,Compound 125

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-(2-amino-2-methyl-propyl)carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 379.

Example 126 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 126

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewas synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate. LCMS (ESI) (M+H) 427.

Example 127 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamate,Compound 127

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.LCMS (ESI) (M+H) 427.

Example 128 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 128

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 369 (M+H).

Example 129 Synthesis of Compound 129

Chloro tricyclic amide, Compound 129, was synthesized using a similarprocedure as that described for the synthesis of chloro tricyclic amide,Compound 119. LCMS (ESI) 251 (M+H).

Example 130 Synthesis of Compound 130

Compound 130 was synthesized by treating chlorotricyclic amine Compound129 with trans-4-aminocyclohexanol using similar experimental conditionsas for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δppm 1.07-1.34 (m, 4H) 1.47-2.05 (m, 10H) 3.09 (m, 1H) 3.51 (d, J=2.91Hz, 2H) 3.57 (m, 1H) 4.50 (br. s., 1H) 6.89 (s, 1H) 6.94-7.05 (m, 1H)8.04 (br. s., 1H) 8.60 (s, 1H) 9.00 (br. s., 1H).

Example 131 Synthesis of benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamate,Compound 131

BenzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine with benzylN-[1-(aminomethyl)propyl]carbamate using similar experimental conditionsas described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 413.

Example 132 Synthesis of benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamate,Compound 132

BenzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewas prepared by treating benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]-carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamateLCMS (ESI) (M+H) 461.

Example 133 Synthesis of benzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamate,Compound 133

BenzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamatewas synthesized by treating benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) (M+H) 461.

Example 134 Synthesis of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 134

7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 403 (M+H).

Example 135 Synthesis of Compound 135

To a solution of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid in dichloromethane was added HBr, the reaction was stirred at 45degrees for 3 hrs. After concentration, 2N NaOH was added to basify(pH=8.0) the reaction followed by the addition of THF (20 mL). Boc₂O wasthen added (1.2 eq) and the reacation was stirred for 16 hrs. To thecrude reaction mixture was then added ethyl acetate (100 mL) and water(50 mL) and the organic phase was separated, dried (magnesium sulfate)and then concentrated under vacuum. To the crude product was addeddichloromethane (30 mL) followed by DIC and DMAP. After stirring for 2hrs, TFA was added and the contents stirred for an hour. The solventswere evaporated under vacuum and the residue basified with satd. NaHCO₃.Ethyl acetate was then added and the organic layer separated, dried(magnesium sulfate) and then concentrared under vacuum. Columchromatography with hexane/ethyl acetate (0-100%) afforded the desiredchlorotricyclic core, Compound 135. LCMS (ESI) 251 (M+H).

Example 136 Synthesis of Compound 136

Compound 136 was synthesized by treating chlorotricyclic amine, Compound135, with trans-4-aminocyclohexanol using similar experimentalconditions as for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.80-0.95 (m, 3H) 1.35-1.92 (m, 10H) 3.66 (br. m., 3H)4.17 (br. s., 2H) 4.47 (br. s., 1H) 6.85 (s, 1H) 6.96 (br. s., 1H) 8.15(br. s., 1H) 8.62 (br. s., 1H).

Example 137 Synthesis of tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamate,Compound 137

tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[1-(aminomethyl)cyclopentyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 138 Synthesis of tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamate,Compound 138

tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewith with 3,3-diethoxyprop-1-yne in the presence of a catalyst such asPddba using similar experimental conditions as described for thesynthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate LCMS (ESI) 453 (M+H).

Example 139 Synthesis of tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamate,Compound 139

tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 453 (M+H).

Example 140 Synthesis of7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 140

7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 141 Synthesis of Compound 141

Chlorotricyclic core Compound 141 was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide Compound 119. LCMS (ESI) 277 (M+H).

Example 142 Synthesis of Compound 142

Compound 142 was synthesized by treating chlorotricyclic amine, Compound141, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.08-1.32 (m, 8H) 1.60-2.09 (m, 8H) 3.03-3.17 (m, 1H)3.35 (s, 2H) 3.54-3.62 (m, 1H) 4.51 (d, J=4.39 Hz, 1H) 6.88 (s, 1H) 6.96(br. s., 1H) 8.07 (br. s., 1H) 8.58 (s, 1H).

Example 143 Synthesis of tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate,Compound 143

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[(1-aminocyclopentyl)methyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 144 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 144

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate.

LCMS (ESI) 453 (M+H).

Example 145 Synthesis of tert-butylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamate,Compound 145

tert-ButylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 4534 (M+H).

Example 146 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6carboxylicacid, Compound 146

7-[2-(tert-Butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 147 Synthesis of Compound 147

Chloro tricyclic amide, Compound 147 was synthesized using a similarexperimental procedure as that described for the chloro tricyclic amide,Compound 119. LCMS (ESI) 277 (M+H).

Example 148 Synthesis of Compound 148

Compound 148 was synthesized by treating chlorotricyclic amine, Compound147, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.06-1.35 (m, 8H) 1.45-1.95 (m, 8H) 3.10 (m, 1H) 3.58(br. s., 2H) 3.95 (br. s., 1H) 4.49 (br. s., 1H) 6.84 (s, 1H) 6.85-6.93(m, 1H) 8.29 (s, 1H) 8.61 (br. s., 1H).

Example 149 Synthesis of Compound 149

Step 1: Compound 59 is Boc protected according to the method of A.Sarkar et al. (JOC, 2011, 76, 7132-7140).Step 2: Boc-protected Compound 59 is treated with 5 mol % NiCl₂(Ph₃)₂,0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, inDMI under CO₂ (1 atm) at 25° C. for 20 hours to convert the aryl halidederivative into the carboxylic acid.Step 3: The carboxylic acid from Step 2 is converted to thecorresponding acid chloride using standard conditions.Step 4: The acid chloride from Step 3 is reacted with N-methylpiperazine to generate the corresponding amide.Step 5: The amide from Step 4 is deprotected using trifluoroacetic acidin methylene chloride to generate the target compound. Compound 149 ispurified by silica gel column chromatography eluting with adichloromethane-methanol gradient to provide Compound 149.

Each of Compounds 119 through 147 and corresponding compounds withvarious R⁸, R¹ and Z definitions may be reacted with sodium hydride andan alkyl halide or other halide to insert the desired R substitutionprior to reaction with an amine, such as described above for thesynthesis of Compound 120, to produce the desired product of Formulae I,II, III, IV, or V.

Example 150

CDK4/6 Inhibition In Vitro Assay

Selected CDK4/6 inhibitor compounds disclosed herein were tested inCDK4/cyclinD1, CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn(Santa Clara, Calif.) to determine their inhibitory effect on theseCDKs. The assays were performed using microfluidic kinase detectiontechnology (Caliper Assay Platform). The compounds were tested in12-point dose-response format in singlicate at Km for ATP.Phosphoacceptor substrate peptide concentration used was 1 μM for allassays and Staurosporine was used as the reference compound for allassays. Specifics of each assay are as described below:

CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM;Incubation time: 3 hr.

CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM;Incubation time: 1 hr.

CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM;Incubation time: 10 hr.

The inhibitory IC₅₀ values for the compounds in Table 1 for CDK4/CycD1,CDK2/CycE, CDK2/CycA, as well as fold selectivity are presented in Table2.

TABLE 2 Selective Inhibition of CDK4 Fold Fold CDK4/ CDK2/ SelectivityCDK2/ Selectivity CycD1 CycE (CDK2/ CycA (CDK2/ Struc- IC₅₀ IC₅₀ CycE/IC₅₀ CycA/ ture [nM] [nM] CDK4) [nM] CDK4) A 4.2 6350 1516 3160 754 B0.4 3040 6862 1890 4266 C 1.4 1920 1333 616 428 D 0.9 3480 3779 15001629 E 1 695 688 204 202 F 1.5 628 419 190 127 G 1.5 2580 1767 646 442 H1.5 1520 1013 377 251 I 2 2120 1065 1130 568 J 0.7 5110 7707 4340 6546 K1 1070 1019 738 703 L 5.7 4530 789 1490 260 M 2.3 2280 1004 1410 621 N 11500 1500 ND ND O 2.5 41410 1636 3150 1245 P 3.3 3560 1085 1010 308 Q0.6 1080 1722 3030 4833 R 0.5 1920 3918 1360 2776 S 1.7 1250 718 342 197T 0.8 1660 2022 1670 2034 U 0.7 1460 2229 857 1308 V 2.9 3500 1224 2130745 W 2.7 3970 1481 539 201 X 0.9 11600 12975 1840 2058 Y 2.5 124 50 6125 Z 3.2 3710 1174 647 205 AA 0.5 6100 13319 4630 10109 BB 0.8 1680 2017502 603 CC 1.6 1250 791 755 478 DD 1.9 9620 5200 8360 4519 EE 3.8 1660432 1110 289 FF 1.2 4620 3949 1400 1197 GG 1 3580 3377 1510 1425 HH 1.71280 766 265 159 II 2 367 184 239 120 JJ 1.4 288 204 ND ND KK 2.3 1760762 915 396 LL 2 202 103 108 55 MM 1.8 3390 1863 597 328 NN 3.7 47001274 1560 423 OO 9 3980 442 570 63 PP 3.1 3600 1146 3090 984 QQ 4.1 3060746 2570 627 RR 1.2 1580 1374 693 603 SS 0.8 1460 1865 1390 1775 TT 0.81260 1550 596 733 UU 7.3 3960 542 ND ND VV 3.3 2630 809 789 243 WW 0.71350 204 ND ND XX 1.3 7300 5615 6290 4838 YY 4.6 6900 1490 ND ND ZZ 10.59960 949 ND ND AAA 2.3 6010 2591 2130 918 BBB 2.8 187 68 85 31 CCC 22170 1074 457 226 DDD 9.5 9350 986 ND ND EEE 0.2 2950 1266 943 405 FFF4.7 4540 966 1370 291 GGG 13.7 7610 555 ND ND HHH 6.8 2840 419 ND ND III6 3770 626 ND ND JJJ 3.2 5200 1620 2830 882 KKK 1.3 291 231 87.3 69 LLL3.2 1620 509 4530 1425 MMM 3.2 1890 600 990 314 NNN 1.4 2930 2154 1010743 OOO 2.4 393 164 203 85 PPP 0.8 16500 21263 2640 3402 QQQ 10.5 111001057 ND ND RRR 2.6 4500 1758 ND ND SSS 2 2280 1112 1880 917 TTT 3.4 3030899 ND ND UUU 18 16460 914 ND ND VVV 7.4 4380 589 ND ND WWW 18.5 2500135 ND ND XXX 11.4 6620 581 ND ND

To further characterize its kinase activity, Compound T was screenedagainst 456 (395 non-mutant) kinases using DiscoveRx's KINOMEscan™profiling service. The compound was screened using a singleconcentration of 1000 nM (>1000 times the IC50 on CDK4). Results fromthis screen confirmed the high potency against CDK4 and high selectivityversus CDK2. Additionally, the kinome profiling showed that Compound Twas relatively selective for CDK4 and CDK6 compared to the other kinasestested. Specifically, when using an inhibitory threshold of 65%, 90%, or99%, Compound T inhibited 92 (23.3%), 31 (7.8%) or 6 (1.5%) of 395non-mutant kinases respectively.

In addition to CDK4 kinase activity, several compounds were also testedagainst CDK6 kinase activity. The results of the CDK6/CycD3 kinaseassays, along with the CDK4/cyclinD1, CDK2/CycA and CDK2/cyclinE kinaseassays, are shown for PD0332991 (Reference) and the compounds T, Q, GG,and U in Table 3. The IC₅₀ of 10 nM for CDK4/cyclinD1 and 10 uM forCDK12/CyclinE agrees well with previously published reports forPD0332991 (Fry et al. Molecular Cancer Therapeutics (2004)3(11)1427-1437; Toogood et al. Journal of Medicinal Chemistry (2005) 48,2388-2406). Compounds T, Q, GG, and U are more potent (lower IC₅₀) withrespect to the reference compound (PD0332991) and demonstrate a higherfold selectivity with respect to the reference compound (CDK2/CycE IC₅₀divided by CDK4/CycD1 IC₅₀).

TABLE 3 Inhibition of CDK kinases by Compounds T, Q, GG, and U CDK4/CDK2/ Fold CDK2/ CDK6/ CycD1 CycE Selectivity CycA CycD3 IC₅₀ IC₅₀ CDK2/IC₅₀ IC50 Formula (nM) (uM) CDK4 (uM) (nM) PD0332991 10 10 1000 Not de-Not de- Reference termined termined Compound T 0.821 1.66 2022 1.67 5.64Compound Q 0.627 1.08 1722 3.03 4.38 Compound GG 1.060 3.58 3377 1.514.70 Compound U 0.655 1.46 2229 .857 5.99

Example 151

G1 Arrest (Cellular G1 and S-Phase) Assay

To show that CDK4/6 inhibitors described herein, when administeredalone, do not have an inhibitory affect in Rb-negative (i.e.,CDK-replication independent) cancers, CDK4/6 inhibitors wereadministered to various Rb-positive and Rb-negative cells and thecellular fractions in various stages of the cell cycle followingtreatment was assessed.

HS68 cells (human skin fibroblast cell line (Rb-positive)) were stainedwith propidium iodide staining solution and run on Dako Cyan FlowCytometer. The fraction of cells in G0-G1 DNA cell cycle versus thefraction in S-phase DNA cell cycle was determined using FlowJo 7.2.2analysis.

The compounds listed in Table 1 were tested for their ability to arrestHS68 cells at the G1 phase of the cell cycle. From the results of thecellular G1 arrest assay, the range of the inhibitory EC₅₀ valuesnecessary for G1 arrest of HS68 cells was from 22 nM to 1500 nM (seecolumn titled “Cellular G1 Arrest EC₅₀” in Table 4).

Example 152

Inhibition of Cellular Proliferation

Further cellular proliferation assays were conducted using the followingcancer cell lines: MCF7 (breast adenocarcinoma—Rb-positive), ZR-75-1(breast ductal carcinoma—Rb-positive), H69 (human small cell lungcancer—Rb-negative) cells, or A2058 (human metastatic melanomacells—Rb-negative). These cells were seeded in Costar (Tewksbury, Mass.)3093 96 well tissue culture treated white walled/clear bottom plates.Cells were treated with the compounds of Table 1 as nine point doseresponse dilution series from 10 uM to 1 nM. Cells were exposed tocompounds and then cell viability was determined after either four (H69)or six (MCF7, ZR75-1, A2058) days as indicated using the CellTiter-Glo®luminescent cell viability assay (CTG; Promega, Madison, Wis., UnitedStates of America) following the manufacturer's recommendations. Plateswere read on BioTek (Winooski, Vt.) Syngergy2 multi-mode plate reader.The Relative Light Units (RLU) were plotted as a result of variablemolar concentration and data was analyzed using Graphpad (LaJolla,Californaia) Prism 5 statistical software to determine the EC₅₀ for eachcompound.

The results of the cellular inhibition assays for the two Rb-positivebreast cancer cell lines (MCF7 and ZR75-1) are shown in Table 4. Therange of the inhibitory EC₅₀ values necessary for inhibition of MCF7breast cancer cell proliferation was 28 nM to 257 nM. The range of theinhibitory EC₅₀ values necessary for inhibition of ZR75-1 breast cancercell proliferation was 24 nM to 581 nM.

In addition to breast cancer cell lines, a number of the compoundsdisclosed herein were also evaluated against a small cell lung cancercell line (H69) and a human metastatic melanoma cell line (A2058), twoRb-negative cell lines. The results of these cellular inhibition assaysare shown in Table 4. The range of the inhibitory EC₅₀ values necessaryfor inhibition of H69 small cell lung cancer cells was 2040 nM to >3000nM. The range of the inhibitory EC₅₀ values necessary for inhibition ofA2058 malignant melanoma cell proliferation was 1313 nM to >3000 nM. Incontrast to the significant inhibition seen on the two Rb-positivebreast cancer cell lines, it was found that the compounds tested werenot significantly effective at inhibiting proliferation of theRb-negative small cell lung cancer or melanoma cells.

TABLE 4 Inhibition of Cancer Cell Proliferation Cellular MCF7 ZR75-1 H69A2058 G1 Arrest Cellular Cellular Cellular Cellular Struc- EC₅₀ EC₅₀EC₅₀ EC₅₀ EC₅₀ ture (nM) [nM] [nM] [nM] [nM] A 110 75 44 >3000 ND B 90201  245  ND ND C 95 88 73 ND ND D 50 57 46  2911  1670 E 75 53 62  2580 1371 F 175 ND ND ND ND G 175 ND ND ND ND H 85 85 120   2040  1313 I 8061 40  2950  1062 J 110 70 82 >3000 >3000 K 28 43 ND >3000  1787 L 65506  ND  2161 >3000 M 100 ND ND ND ND N 25 28 24 >3000  1444 O 40 5629 >3000  2668 P 30 60 43 >3000 >3000 Q 100 49 35 >3000  2610 R 70 3650 >3000  2632 S 150 76 ND >3000 >3000 T 100 49 36 >3000 >3000 U 25 7059 >3000 >3000 V 70 50 29 >3000  1353 W 160 294  ND >3000 >3000 X 65 NDND >3000 >3000 Y 350 ND ND ND ND Z 110 141  54 ND ND AA 70 47 47 >3000ND BB 75 ND ND  2943  1635 CC 90 50 38 >3000 >3000 DD 100 ND ND ND ND EE125 216  203  ND ND FF 80 140  ND ND ND GG 80 52 62  2920  2691 HH 110ND ND ND ND II 40 94 33 >3000 >3000 JJ 90 122  ND >3000 >3000 KK 22 333 ND  2421  1379 LL 125 96 ND >3000 >3000 MM 100 73 77 >3000 >3000 NN 110ND ND ND ND OO 95 120  229  >3000 >3000 PP 100 164  66 ND ND QQ 120 NDND >3000 >3000 RR 90 72 ND  2888  1617 SS 80 94 53  2948  1658 TT 75 NDND ND ND UU 300 ND ND ND ND VV 200 ND ND ND ND WW 400 ND ND ND ND XX 225ND ND ND ND YY 175 257  581  ND ND ZZ 500 ND ND ND ND AAA 275 320 ND >3000 >3000 BBB 230 123  ND >3000 >3000 CCC 250 ND ND ND ND DDD 350ND ND ND ND EEE 250 453  ND >3000 >3000 FFF 650 ND ND ND ND GGG 350 NDND ND ND HHH 250 ND ND ND ND III 250 ND ND ND ND JJJ 240 ND ND ND ND KKK190 ND ND ND ND LLL 250 ND ND ND ND MMM 200 134  141  >3000 >3000 NNN210 ND ND ND ND OOO 200 138  ND >3000 >3000 PPP 275 ND ND ND ND QQQ 500ND ND ND ND RRR 400 ND ND ND ND SSS 1500 ND ND ND ND TTT 350 ND ND ND NDUUU 300 ND ND ND ND VVV 300 ND ND ND ND WWW 300 ND ND ND ND XXX 300 NDND ND ND

Example 153

Pharmacokinetic and Pharmacodynamic Properties of Active Compounds

Compounds of the present invention demonstrate good pharmacokinetic andpharmacodynamic properties. Compound T, Q, GG, and U were dosed to miceat 30 mg/kg by oral gavage or 10 mg/kg by intravenous injection. Bloodsamples were taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours postdosing and the plasma concentration of compound T, Q, GG, or U weredetermined by HPLC. Compound T, GG, and U were demonstrated to haveexcellent oral pharmacokinetic and pharmacodynamic properties as shownin Table 5. This includes very high oral bioavailability (F (%)) of 52%to 80% and a plasma half-life of 3 to 5 hours following oraladministration. Compound T, Q, GG, and U were demonstrated to haveexcellent pharmacokinetic and pharmacodynamic properties when deliveredby intravenous administration.

TABLE 5 Pharmacokinetic and pharmacodynamic properties of activecompounds Com- Com- Com- Com- Mouse PK pound T pound Q pound GG pound UCL (mL/min/kg) 35 44 82 52 Vss (L/kg) 2.7 5.2 7.5 3.4 t_(1/2) (h) p.o. 50.8 3.5 3 AUC_(0-inf) (uM * h) i.v. 1.3 0.95 1.1 0.76 AUC (uM * h) p.o.2.9 0.15 1.9 3.3 C_(max) (uM) p.o. 2.5 0.16 1.9 4.2 T_(max) (h) p.o. 10.5 1 0.5 F (%) 80 2 52 67

Example 154

Cell Cycle Arrest by CDK 4/6 Inhibitors in CDK4/6-Dependent Cells

To test the ability of the disclosed CDK4/6 inhibitors to induce a cleanG1-arrest, a cell based screening method was used consisting of twoRb-positive (tHS68 and WM2664) and one Rb-negative (A2058) cell line.Twenty-four hours after plating, each cell line was treated withCompound T in a dose dependent manner for 24 hours. At the conclusion ofthe experiment, cells were harvested, fixed, and stained with propidiumiodide (a DNA intercalator), which fluoresces strongly red (emissionmaximum 637 nm) when excited by 488 nm light. Samples were run on DakoCyan flow cytometer and >10,000 events were collected for each sample.Data were analyzed using FlowJo 2.2 software developed by TreeStar, Inc.

In FIG. 9A, results show that CDK 4/6 inhibitor Compound T induces arobust G1 cell cycle arrest, as nearly all cells are found in the G0-G1phase upon treatment with increasing amounts of Compound T. In FIG. 9A,the results show that in Rb-positive cell lines, Compound T induced arobust G1 cell cycle arrest with an EC₅₀ of 80 nM in tHS68 cells with acorresponding reduction in S-phase ranging from 28% at baseline to 6% atthe highest concentration shown. Upon treatment with Compound T (300nM), there was a similar reduction in the S-phase population and anincrease in G1-arrested cells in both Rb-positive cell lines (tHS68(Compare FIGS. 9B and 9E) and WM2664 (Compare FIGS. 9C and 9F)), but notin the Rb-negative (A2058; Compare FIGS. 9D and 9G) cell line. TheRb-negative cell line shows no effect in the presence of inhibitor.

Example 155

CDK 4/6 Inhibitor Compounds Inhibit Phosphorylation of RB

The CDK4/6-cyclin D complex is essential for progression from G1 to theS-phase of the DNA cell cycle. This complex phosphorylates theretinoblastoma tumor suppressor protein (Rb). To demonstrate the impactof CDK4/6 inhibition on Rb phosphorylation (pRb), CDK 4/6 inhibitorCompound T was exposed to three cell lines: two Rb-positive (tHS68,WM2664) and one Rb-negative (A2058). Twenty four hours after seeding,cells were treated with Compound T at 300 nM final concentration for 4,8, 16, and 24 hours. Samples were lysed and protein was assayed bywestern blot analysis. Rb phosphorylation was measured at two sitestargeted by the CDK4/6-cyclin D complex, Ser780 and Ser807/811 usingspecies specific antibodies. Results demonstrate that Compound T blocksRb phosphorylation in Rb-positive cell lines by 16 hours post exposure,while having no effect on Rb-negative cells (FIG. 10).

Example 156

CDK 4/6 Inhibitor Compounds Show Enhanced Efficacy Against CDK4/6Independent Tumors in Combination with a Topoisomerase I Inhibitor

The potential of the disclosed CDK 4/6 inhibitors to have advantageous,additive, or synergistic effects in combination with topoisomerase Iinhibitors was examined in an Rb-negative tumor model. An Rb-negativemodel of small cell lung cancer was used in the following example.Briefly, xenografts of National Cancer Institute (NCI) H69 cells weregrafted onto the mice. H69 cells are human small cell lung cancer cellsand are Rb-negative cells.

Mice were enrolled in therapy studies when tumors reached a standardsize (50-60 mm³) that permitted easy serial assessment. Tumor-bearingmice were treated with vehicle control (N=8), carboplatin/etoposide(N=8), Compound T/carboplatin/etoposide (N=8), topotecan (N=8), orCompond T/topotecan (N=7). Carboplatin and etoposide were administeredat 40 mg/kg and 4 mg/kg, respectively, and were administered byintraperitoneal injection twice per week for 3 weeks. Topotecan wasadministered at 0.6 mg/kg by intraperitoneal injection once daily for 5days. Compound T was administered at 100 mg/kg by po, qd×5×4. On dayswhen mice were treated with chemotherapeutic agents, Compound T wasadministered 30 minutes prior to each administration ofcarboplatin/etoposide or topotecan. Tumor size was examined twice perweek for up to nine weeks.

As shown in FIG. 11, treatment of NCI-H69 xenograft mice with the CDk4/6 inhibitor Compound T alone had very little effect of slowing tumorgrowth in the Rb-negative tumor model. When Compound T was administeredin combination with the chemotherapeutic agents carboplatin andetoposide, there was no effect on tumor growth when compared to the useof carboplatin and etoposide without Compound T. While Compound T didnot appear to show any synergistic effects in combination withcarboplatin and etoposide, advantageously when Compound T wasadministered in combination with a topoisomerase I inhibitor, in thiscase topotecan, there was a significant decrease in tumor size in theH69 xenograft mouse model.

Example 157

CDK 4/6 Inhibitors Show Enhanced Efficacy Against CDK4/6 IndependentTumors in Combination with Toposiomerase I Inhibitors at Reduced CDK 4/6Dosages

The potential of the disclosed CDK 4/6 inhibitors to have advantageous,additive, or synergistic effects in combination with a topoisomerase Iinhibitor compound, in this instance topotecan, was further examined inan Rb-negative tumor model. An Rb-negative model of small cell lungcancer was used in the following example. Briefly, xenografts ofNational Cancer Institute (NCI) H69 cells were grafted onto the mice(Athymic Nude (Crl:NU(NCr)-Foxn1^(nu)); 6-12 week old females) using theSouth Texas Accelerated Research Therapeutics (START) Cell-BasedXenograft (START-CBX) tumor model. H69 cells are human small cell lungcancer cells and are Rb-negative.

Data collected from the efficacy study included animal weights,observations and tumor dimensions. This information was used todetermine agent tolerability based on weight change and grossphysiologic changes and anticancer activity based on tumor growth delayor regression. The designated endpoint for this study was an individualtumor volume of 1 cm³ or 60 days following treatment initiation.

Tumor fragments from NCI-H69 were harvested from host animals andimplanted into immune-deficient mice and the study initiated at a meantumor volume of approximately 150-250 mm³. Tumor-bearing mice weretreated with Compound T vehicle and topotecan vehicle (Group 1; N=7),Compound T (100 mg/kg) and topotecan vehicle (Group 2; N=7), Compound T(100 mg/kg) and topotecan (0.6 mg/kg) (Group 3; N=7), Compound T (50mg/kg) and topotecan (0.6 mg/kg) (Group 4; N=7), Compound T (10 mg/kg)and topotecan (0.6 mg/kg) (Group 5; N=7) or Compound T vehicle andtopotecan (0.6 mg/kg) (Group 6; N=7).

Importantly, the advantageous anti-tumor effects seen with the use ofCDK4/6 inhibitor compounds described herein in combination withtopoisomerase I inhibitors occurred at all doses of CDK4/6 inhibitor—100mg/kg, 50 mg/kg, and 10 mg/kg. Thus, an advantageous anti-tumor effectcan be achieved with low doses of CDK4/6 inhibitors described herein,for example about 10 mg/kg.

Additional details of the study are outlined in Table 6 below. On dayswhen mice were treated with chemotherapeutic agents, Compound T wasadministered 30 minutes prior to topotecan. Tumor size was examinedtwice per week and the endpoint for this study was Day 59.

TABLE 6 Detailed Study Outline Animals Dose Total Treatment Group (N)Treatment (mg/kg) ROA/Schedule Doses Days Endpoint 1 7 Compound T —PO/qd × 5 × 4 15 0-4, 7-11, 21 Vehicle 14-18 Topotecan Vehicle — IP/qd ×5 × 4 15 0-4, 7-11, 14-18 2 7 Compound T 100 PO/qd × 5 × 4 19 0-4, 7-11,24 14-18, 21-23 Topotecan Vehicle — IP/qd × 5 × 4 19 0-4, 7-11, 14-18,21-23 3 7 Compound T 100 PO/qd × 5 × 4 20 0-4, 7-11, 59 14-18, 21-24Topotecan 0.6 IP/qd × 5 × 4 20 0-4, 7-11, 14-18, 21-24 4 7 Compound T 50PO/qd × 5 × 4 20 0-4, 7-11, 59 14-18, 21-24 Topotecan 0.6 IP/qd × 5 × 420 0-4, 7-11, 14-18, 21-24 5 7 Compound T 10 PO/qd × 5 × 4 20 0-4, 7-11,59 14-18, 21-24 Topotecan 0.6 IP/qd × 5 × 4 20 0-4, 7-11, 14-18, 21-24 67 Compound T — PO/qd × 5 × 4 20 0-4, 7-11, 59 Vehicle 14-18, 21-24Topotecan 0.6 IP/qd × 5 × 4 20 0-4, 7-11, 14-18, 21-24

As shown in FIG. 12, the tumor doubling time of the NCI-H69 xenograftswas 5.8 days. As shown in FIG. 13, Compound T, administered alone orthirty minutes before administration of a topoisomerase Iinhibitor-topotecan, was well tolerated with no additive weight loss ortoxicity. As shown in FIG. 14, there was no tumor burden associated withthis model based on the percent weight gain versus Day 0 in controlgroup animals.

As shown in FIG. 15, mice treated with topotecan or topotecan/Compound Tsurvived significantly longer than mice treated with vehicle or CompoundT only. The combination of topotecan with 100 mg/kg Compound T or 50mg/kg Compound T led to increased survival rates in comparison totopotecan alone.

As shown in FIG. 16, Compound T alone was inactive toward NCI-H69xenografts, while topotecan alone was active and reduced tumor volumes(p<0.0001; vs. vehicle control). Additionally, the combination oftopotecan with each concentration of Compound T tested (100 mg/kg, 50mg/kg, or 10 mg/kg) led to a reduction in tumor volume in comparison totreatment with vehicle (p<0.0001). The results of the experimentsconducted in FIG. 16 are summarized in Table 7.

TABLE 7 Agent Efficacy and Tumor Volume Data MTTE ± *Signif- PR Group SDT − C *p-value icant LTS (%) CR TFS 1 18 ± 3 — — — 0 0 0 0 2 14 ± 4 <0 0.8813 N 0 0 0 0 3 55 ± 3 37 <0.0001 Y 3 4 0 0 4 59 ± 4 41 <0.0001 Y 35 0 0 5 55 ± 4 37 <0.0001 Y 1 3 0 0 6 52 ± 7 34 <0.0001 Y 1 0 0 0*Versus Vehicle Control Group 1 = Compound T vehicle and topotecanvehicle Group 2 = Compound T (100 mg/kg) and topotecan vehicle Group 3 =Compound T (100 mg/kg) and topotecan (0.6 mg/kg) Group 4 = Compound T(50 mg/kg) and topotecan (0.6 mg/kg) Group 5 = Compound T (10 mg/kg) andtopotecan (0.6 mg/kg) Group 6 = Compound T vehicle and topotecan (0.6mg/kg) MTTE = Median timepoint in days for an individual mouse or groupto reach a designated tumor volume or time endpoint, Mean ± SD used. T −C = Difference in days between MTTE of treatment and control groups. LTS= Individual tumor volume (TV) < volume endpoint at study time endpoint.PR (%) = Tumor volume (TV) ≥50% regression versus Day 0 for twoconsecutive measurements over a period of ≥7 days during or at studycompletion. CR = Tumor volume (TV) <15 mm³ for two consecutivemeasurements over a period of ≥7 days during or at study completion. TFS= A designated complete responder at study endpoint; TFS animals areexcluded from efficacy calculations.

This specification has been described with reference to embodiments ofthe invention. The invention has been described with reference toassorted embodiments, which are illustrated by the accompanyingExamples. The invention can, however, be embodied in different forms andshould not be construed as limited to the embodiments set forth herein.Given the teaching herein, one of ordinary skill in the art will be ableto modify the invention for a desired purpose and such variations areconsidered within the scope of the invention.

The invention claimed is:
 1. A method for treating a human havingRb-negative small cell lung cancer comprising: a. administering to thehuman an effective amount of Cyclin Dependent Kinase (CDK) 4/6 inhibitorcompound of the formula:

or a pharmaceutically acceptable salt therein, and, b. administering tothe human an effective amount of belotecan, wherein the belotecan isadministered about 24 hours or less after the administration of the CDK4/6 inhibitor.
 2. The method of claim 1, wherein belotecan isadministered about 4 hours or less after the administration of the CDK4/6 inhibitor.
 3. The method of claim 1, wherein belotecan isadministered about 30 minutes or less after the administration of theCDK 4/6 inhibitor.